Formulations, methods, kits, and dosage forms for treating atopic dermatitis and for improved stability of an active pharmaceutical ingredient

ABSTRACT

Embodiments of the disclosure relate generally to formulations, methods of treatment, kits, and dosage forms for treating inflammatory disorders, including atopic dermatitis, or cancer, the formulations comprising an active pharmaceutical ingredient. The formulation provided comprises granules, wherein the granules comprise: micronized active ingredient; one or more granulation binders; one or more fillers; one or more disintegrants; and one or more antioxidants. In one embodiment, the methods of treatment include orally administering the active ingredient to a subject suffering from atopic dermatitis, where the active ingredient is in an amount of about 20 mg to about 80 mg.

TECHNICAL FIELD

Embodiments of the disclosure relate generally to formulations, methods,kits, and dosage forms for treating atopic dermatitis and for improvedstability of an active pharmaceutical ingredient. In one embodiment, theformulations, methods, kits and dosage forms comprise administering theactive pharmaceutical ingredient with improved stability and can be usedfor the treatment of inflammatory disorders or cancers, or for thetreatment of atopic dermatitis.

BACKGROUND

Protein kinases constitute a large family of structurally relatedenzymes that are responsible for the control of a variety of signaltransduction processes within cells. Almost all kinases contain asimilar 250 to 300 amino acid catalytic domain. The kinases can becategorized into families by the substrates they phosphorylate.

JAK (Janus kinase, including JAK1, JAK2, JAK3 and TYK2) is a family ofintracellular non-receptor tyrosine kinases. JAK is expressed inhematopoietic cells and abundantly in primary leukemic cells fromchildren with acute lymphoblastic leukemia. The downstream substrates ofJAK include the signal tranducer activator of transcription (STAT)proteins. STAT proteins function both as signaling molecules andtranscription factors and ultimately bind to specific DNA sequencespresent in the promoters of cytokine-responsive genes. JAK/STATsignaling has been implicated in the mediation of many abnormal immuneresponses such as allergies, asthma, autoimmune diseases such astransplant (allograft) rejection, rheumatoid arthritis, amyotrophiclateral sclerosis and multiple sclerosis, as well as in solid andhematologic malignancies such as leukemia and lymphomas.

Spleen tyrosine kinase (syk) is a member of the syk family of proteintyrosine kinases and plays a crucial role in inflammatory and allergicresponses. Syk triggers IgE and IgG receptor mediated signaling in mastcells, basophils, and macrophages leading to degranulation and cytokinerelease.

Immunoreceptor tyrosine activation motif (ITAM)-mediated signaling hasemerged as a primary event in signaling pathways responsible for humanpathologies. ITAM-mediated signaling is responsible for relayingactivation signals initiated at classical immune receptors such asT-cell receptors, B-cell receptors, and Fc receptors in immune cells andat GPVI and FcγRIIa in platelets to downstream intracellular moleculessuch as Syk.

The binding of a ligand to an ITAM-containing receptor triggerssignaling events which allows for the recruitment of proteins from afamily of nonreceptor tyrosine kinases called the Src family. Thesekinases phosphorylate tyrosine residues within the ITAM sequence, aregion with which the tandem SH2 domains on either Syk or ZAP-70interact. The interaction of Syk with diphosphorylated ITAM sequencesinduces a conformation change in the kinases that allows for tyrosinephosphorylation of the kinase itself.

Not only do these kinases contribute to normal host defense, they alsoplay roles in the pathogenesis of diseases. Many diseases are associatedwith abnormal cellular responses triggered by protein kinase-mediatedevents. These diseases include autoimmune diseases, inflammatorydiseases, bone diseases, metabolic diseases, neurological andneurodegenerative diseases, cancer, cardiovascular diseases, allergies,asthma, Alzheimer's disease and hormone-related diseases. As aconsequence, there have been substantial efforts in medicinal chemistryto find inhibitors of protein kinases for use as therapeutic agents.There is a need in the art for compounds that are dual inhibitors ofSyk/JAK, as well as for methods for treating conditions that can benefitfrom such inhibition. There is also a need in the art for formulationsof compounds that are dual inhibitors of Syk/JAK, that may be utilizedin methods for treating conditions that can benefit from suchinhibition. Such formulations should optimize the efficacy of Syk/JAKdual inhibitor compounds and should exhibit high levels of stability.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. It ischaracterized by dry scaly skin, erythema, lesions with oozing andcrusting, excoriations due to itch, and lichenification. The skinconditions are accompanied by intense pruritus that poses a significantburden to subjects and their quality of life. Up to 3% of adults haveatopic dermatitis and between 15-25% of children. Onset is in earlychildhood in approximately 85% of cases but onset can occur laterincluding during adulthood.

Spleen tyrosine kinase (SYK) and Janus kinase (JAK) are tyrosine kinasesthat play important roles in the pathogenesis of various types ofautoimmune and inflammatory diseases, including atopic dermatitis.Deregulation of SYK has been implicated in different human diseases suchas B-cell malignancies, allergy, asthma, and other inflammatorydisorders. SYK binds to the immune-receptor tyrosine-based activationmotif (ITAM) present in Fcγ-activating receptors and integrins. Bindingof SYK to the ITAM activates downstream signaling events such asactivation of Bruton tyrosine kinase (BTK), eventually leading toincreased release of cytokines, lipid mediators and various proteases.These mediators cause hyper-proliferation of B-cells, inflammation, andtissue or cartilage damage. SYK also plays a critical role in IL-17Rsignaling in keratinocytes. Therefore, inhibition of SYK activityprovides a potential approach for the treatment of various types oflymphomas and inflammatory disorders.

The JAK kinases (JAK1, JAK2, JAK3 and TYK2) are required for thephysiologic signaling through the cytokines and growth factor receptorsthat intrinsically lack kinase activity (12, 13). JAK kinases, uponstimulation with factors such as erythropoietin, granulocyte-macrophagecolony stimulating factor, IL-3, IL-5, thrombopoietin, and growthhormone, phosphorylate signal transducers and activators oftranscription (STAT1-5) family proteins which are translocated to thenucleus and activate various downstream target genes involved incytokine and growth factor response. JAK kinases play a role ininflammatory conditions, particularly those driven by cytokinesincluding atopic dermatitis.

There is a need in the art for methods of treatment using compounds thatare dual inhibitors of Syk/JAK for treating inflammatory disorders, suchas atopic dermatitis.

SUMMARY

In one embodiment, the present disclosure relates to formulations,methods, kits, and dosage forms for treating conditions related to theinhibition of Syk/JAK, such as such as inflammatory disorders orcancers, characterized by the presence of solid tumors, particularlymelanoma, colon cancer, non-small cell lung cancer, bladder cancer andbreast cancer and/or the following cancers: prostate, head, neck, eye,mouth, throat, esophagus, bronchus, larynx, pharynx, chest, bone,rectum, stomach, uterus, cervix, ovaries, vagina, testicles, skin,thyroid, blood, lymph nodes, kidney, liver, intestines, pancreas, brain,central nervous system, adrenal gland, skin or a leukemia and/orlymphoma.

In one embodiment, the present disclosure relates to formulations,methods, kits, and dosage forms for treating conditions related to theinhibition of Syk/JAK, such as inflammatory disorders including atopicdermatitis.

In an embodiment, the pharmaceutical formulations described hereincomprise granules, wherein the granules comprise a micronized activeingredient, one or more granulation binders, one or more fillers, one ormore disintegrants and one or more antioxidants. The formulations mayfurther comprise extragranular components. The active ingredient can bein the amount of about 20 mg to about 80 mg. The formulations describedherein can be administered to a subject once daily for a short-term orlong-term.

The active ingredient may comprise a compound of Formula (I) shownbelow, or a pharmaceutically acceptable salt or prodrug thereof,

wherein R¹ is a 6-membered ring of Formula (II):

wherein R³ is H, OH, C(O)OH, C₁ to C₆ alkyl or (C₁ to C₆) alkyl CN; andwherein R² is a benzene ring of Formula (III):

wherein R₄ is a 6-membered ring of Formula (IV):

wherein R₅ is N or CH and R₆ is a hydroxyl group, methyl group, or ethylgroup, and wherein the formulation has a total API degradation impuritylevel not above about 0.6% of the total active ingredient amount afterstorage at 1 week at 40° C./75% RH in an open container.

In another embodiment, the present disclosure provides a dosage formcomprising a pharmaceutical formulation comprising an active ingredientof formula (I) in a compressed tablet wherein the active ingredient inthe pharmaceutical formulation retains stability after storage for apredetermined time and under predetermined conditions, including in anopen container. In some embodiments, “storage in an open container”means that the container was opened once or twice a day for a givenperiod of time, for example up to four weeks, but was otherwise leftclosed. In one embodiment, the formulation can be used to treat atopicdermatitis. In one embodiment, the active ingredient can be in an amountof about 20 mg to about 80 mg.

In another embodiment, the present disclosure provides a method ofmanufacturing or stabilizing a pharmaceutical formulation. Theformulation can be useful for the treatment of atopic dermatitis. Themethod can comprise the steps of mixing intragranular ingredientscomprising an active ingredient; one or more fillers; one or moredisintegrants; and one or more antioxidants; granulating the mixedintragranular ingredients while adding a solution of 10% w/w of one ormore granulation binders in 99% v/v isopropyl alcohol until granules areformed; and drying and milling the granules to make micronized granules;wherein the active ingredient comprises a compound of Formula (I) or apharmaceutically acceptable salt or prodrug thereof and wherein thepharmaceutical formulation may further comprise extragranularcomponents. In said embodiment, the active ingredient in the formulationretains stability and efficacy for a predetermined time and underpredetermined conditions, including conditions wherein the container maybe opened once or more than once. In one embodiment, the formulation cancomprise an active ingredient in an amount of about 20 mg to about 80mg.

In another embodiment, the present disclosure provides a kit comprisingone or more dosage forms and instructions for administering the dosageforms to a subject, wherein the dosage forms comprise a pharmaceuticalformulation comprising an active ingredient in substantially compressedtablet form optionally combined with extragranular components, whereinthe active ingredient comprises a compound of the formula (I), whereinthe active ingredient in the pharmaceutical formulation retainsstability for a predetermined time and under predetermined conditions.

In another embodiment, the present disclosure provides a method oftreating a condition characterized by dysregulation (e.g., abnormalityor impairment) of Syk/JAK pathways in a subject. In one embodiment, thepresent disclosure provides a method of treating a conditioncharacterized by dysregulation (e.g., abnormality or impairment) ofSyk/JAK2 pathways in a subject. In another embodiment, the presentdisclosure provides methods for treating atopic dermatitis. The methodscan comprise administering to the subject a therapeutically effectiveamount of an active ingredient in one or more dosage forms, wherein thedosage forms comprise a pharmaceutical formulation comprising an activeingredient in granular form in a compressed tablet optionally comprisingone or more extragranular components, wherein the active ingredientcomprises a compound of formula (I), wherein the active ingredientretains stability after storage of the pharmaceutical formulation for apredetermined time and under predetermined conditions. The dosage formscan comprise an active ingredient in the amount of about 20 mg to about80 mg and can be administered to a subject once daily for a short-termperiod or a long-term period.

In another embodiment, the present disclosure provides a pharmaceuticalformulation. The formulation can be useful for treating atopicdermatitis. The formulation can comprise an active ingredient ingranular form in a compressed tablet optionally comprising one or moreextragranular components, wherein the active ingredient comprises activeingredients of the formulations described herein wherein the activeingredient comprises2-(1-(4-((4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile(sometimes referred to herein as “Compound 1”), or wherein the activeingredient comprises2-(1-(4-((4-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile.The active ingredient can be in the amount of about 20 mg to about 80mg.

BRIEF DESCRIPTION OF FIGURES

FIGS. 1A-1B provide a particle size distribution results analysis reportfor the active ingredient prior to micronization. FIG. 1A provides agraph showing the particle size distribution results for the activeingredient prior to micronization. FIG. 1B provides data for theparticle size distribution results for the active ingredient prior tomicronization

FIGS. 2A-2B provide a particle size distribution results analysis reportfor the active ingredient after micronization. FIG. 2A provides a graphshowing the particle size distribution results analysis report for theactive ingredient after micronization. FIG. 2B provides data for theparticle size distribution results analysis report for the activeingredient after micronization.

FIG. 3 provides the 5-D Pruritus Scale.

FIG. 4 provides the Eczema Area and Severity Index (EASI) assessmenttool.

FIG. 5 is a graphical illustration of the study design of Example 3.

FIG. 6 is a graphical illustration of the patient demographics ofExample 3.

FIG. 7A is a graph of the % of subjects to achieve EASI50 over time (Day1 to Day 29) for a placebo and Compound 1 in the doses of 20 mg, 40 mgand 80 mg, as demonstrated in Example 3.

FIG. 7B is a graph of the % of subjects to achieve EASI75 over time (Day1 to Day 29) for a placebo and Compound 1 in the doses of 20 mg, 40 mgand 80 mg, as demonstrated in Example 3.

FIG. 8A is a graph of the % CFB (percentage change from baseline) forEASI (decrease) for the placebo and Compound 1 in the doses of 20 mg, 40mg and 80 mg, as demonstrated in Example 3.

FIG. 8B is a graph of the % CFB for IGA 0-1 (Investigator's GlobalAssessment) for the placebo and Compound 1 in the doses of 20 mg, 40 mgand 80 mg, as demonstrated in Example 3.

FIG. 8C is a graph of the % CFB for BSA (body surface area) (decrease)for the placebo and Compound 1 in the doses of 20 mg, 40 mg and 80 mg,as demonstrated in Example 3.

FIG. 9 is a graph of day 15 plasma concentration for Compound 1 in thedoses of 20 mg, 40 mg and 80 mg, as demonstrated in Example 3.

FIG. 10 is a chart showing the inhibition of JAK and Syk kinase activityby Compound 1, Tofacitinib, Upadacitinib, and Baricitinib, asdemonstrated in Example 3.

FIG. 11 is a chart showing Compound 1's inhibition of JAK/STAT pathwayin T cells stimulated with various cytokines, as demonstrated in Example3.

FIG. 12 is a chart showing Compound 1's inhibition of IL17 mediatedCCL20 release in keratinocytes, as demonstrated in Example 3.

FIG. 13 is a graph showing average weekly change in pruritus (NRS) for aplacebo, and Compound 1 in the doses of 20 mg, 40 mg and 80 mg, asdemonstrated in Example 3.

FIG. 14A is a graph showing improvement in skin thickness for Compound 1in the doses of 20 mg, 40 mg and 80 mg.

FIG. 14B is a graph showing improvement in CD3+ cells for Compound 1 inthe doses of 20 mg, 40 mg and 80 mg.

FIG. 14C is a graph showing improvement in CD11c+ cells for Compound 1in the doses of 20 mg, 40 mg and 80 mg.

FIG. 15 is a chart showing Treatment-Emergent Adverse Events (TEAE), asdemonstrated in Example 3.

FIGS. 16A1-16G2 provide clinical activity, safety and tolerability datafor formulations of the present disclosure.

FIG. 16A1 provides background information and a summary of methods,results and conclusions for formulations of the present disclosure.

FIG. 16A2 provides preclinical study data for formulations of thepresent disclosure.

FIG. 16A3 provides preclinical study data for formulations of thepresent disclosure.

FIG. 16B provides dosing and patient parameters for clinical trial datafor formulations of the present disclosure.

FIG. 16C provides patient demographics and mean pharmacokineticparameters for clinical trial data for formulations of the presentdisclosure.

FIG. 16D provides a graph of the mean plasma concentration of a compoundof the present disclosure over time in preclinical models.

FIG. 16E provides a bar graph of inhibition of inflammation biomarkersby formulations of the present disclosure.

FIG. 16F provides bar graphs showing duration of treatment offormulations of the present disclosure for lymphoma subjects and solidtumor subjects.

FIG. 16G1 provides a description of the efficacy and safety/tolerabilityprofile for a compound of the present disclosure.

FIG. 16G2 provides a summary of the efficacy and safety/tolerabilityprofile for a compound of the present disclosure.

DETAILED DESCRIPTION

The following detailed description is exemplary and explanatory and isintended to provide further explanation of the present disclosuredescribed herein. Other advantages, and novel features will be readilyapparent to one of ordinary skill in the art from the following detaileddescription of the present disclosure.

The present disclosure provides one or more pharmaceutical formulationscomprising an active ingredient in granular form compressed into a soliddosage form such as a tablet, methods of manufacturing suchformulations, kits, methods of treating, and dosage forms wherein theactive ingredient is configured to regulate the Syk/JAK pathway so thatsuch formulations are capable of treating conditions associated withdysregulation in these pathways, including but not limited to, cancersand inflammatory disorders, for example atopic dermatitis.

The present disclosure comprises novel formulations comprisingpyrimido-pyridazinone compounds. The formulations described herein areuseful in treating cancer and inflammatory disorders in patients, forexample atopic dermatitis, by administering one or more of theformulations to patients in need thereof. The formulations describedherein are particularly desirable because of their unexpected superiorstability profiles over a predetermined amount of time and because oftheir efficacy.

In an embodiment, the pharmaceutical formulations described hereincomprise micronized granules, wherein the granules comprise an activeingredient, one or more granulation binders, one or more fillers, one ormore disintegrants and one or more antioxidants. The formulations mayfurther comprise extragranular components.

The active ingredient may comprise a compound of Formula (I) shownbelow, or a pharmaceutically acceptable salt or prodrug thereof,

wherein R¹ is a 6-membered ring of Formula (II):

wherein R³ is H, OH, C(O)OH, C₁ to C₆ alkyl or (C₁ to C₆) alkyl CN; andwherein R² is a benzene ring of Formula (III):

wherein R₄ is a 6-membered ring of Formula (IV):

wherein R₅ is N or CH and R₆ is a hydroxyl group, methyl group, or ethylgroup, and wherein the formulation has a total API degradation impuritylevel not above about 0.6% of the total active ingredient amount afterstorage at 1 week at 40° C./75% RH in an open container.

Compounds of Formula (I) possess one or more chiral centers, and it isspecifically contemplated that each separate enantiomer of compoundscomprising an active ingredient of the disclosure, as well as mixturesof the enantiomers, can be used in the present formulations and methods.As disclosed herein, all chiral, enantiomeric and racemic forms of achemical structure are intended, unless the specific stereochemistry isindicated. It is well known in the art how to prepare optically activeforms of the compounds comprising active ingredients of the presentformulations and methods, such as by resolution of racemic forms or bysynthesis from optically active starting materials.

Active ingredients of the present disclosure can be prepared, forexample, according to the methods disclosed in U.S. Pat. Nos. 8,729,079and 9,382,277, the entire disclosures of which are herein incorporatedby reference. In some embodiments of the disclosure, an activeingredient comprising the pharmaceutical formulation of the disclosurecan be present in at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% w/w.

The active ingredient for use in the present formulations and methodscomprises compounds which regulate the Syk/JAK pathway. The regulatoryactivity of the active ingredients of the disclosure makes thesecompounds useful for manufacturing pharmaceutical formulations, whichcan be used for treating conditions such as inflammatory disorders,including atopic dermatitis, or cancers characterized by the presence ofsolid tumors, particularly melanoma, colon cancer, non-small cell lungcancer, bladder cancer and breast cancer.

In certain embodiments, the active ingredient is2-(1-(4-((4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile.In other embodiments, the active ingredient is2-(1-(4-((4-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile.

The present disclosure thus provides for stable or stabilizedpharmaceutical formulations comprising an active ingredient of thedisclosure as described herein, for example stable or stabilizedformulations comprising one or more compounds of formula (I), orenantiomers, prodrugs, pharmaceutically acceptable salts or free basesthereof. The stability of a formulation according to the presentdisclosure can be determined, for example, by measuring the physicalstate of the active ingredient. In one embodiment, the active ingredientretains stability and efficacy after storage for predetermined times andunder predetermined conditions.

As used herein, the term “substantially granular” means that most of theactive ingredient in the pharmaceutical compositions as describedherein, is in the form of granules, preferably micronized granules,wherein such granules are compressed into a tablet. In certainembodiments, substantially granular means that the granules have aparticle size of less than about 20 microns.

As discussed above, the active ingredient of the present disclosure ismaintained in substantially granular form by combining the activeingredients with one or more stabilizing components. Suitablestabilizing components for use according to the present disclosureinclude one or more granulation binders; one or more fillers; one ormore disintegrants; and one or more antioxidants, as further describedherein.

The method by which the active ingredient and stabilizing component isformulated can also affect stability. For example, mixing intragranularingredients comprising the active ingredient with one or more fillers;one or more disintegrants; and one or more antioxidants; granulating themixed intragranular ingredients while adding a solution of 10% w/w ofone or more granulation binders in 99% v/v isopropyl alcohol untilgranules are formed; and then drying and milling the granules to makemicronized granules results in the formation of granules suitable forincorporating into a compressed tablet and for maintaining stability ofa prolonged period.

In some embodiments, the formulations of the disclosure are stable whensubject to predetermined conditions for predetermined times. Forexample, pharmaceutical formulations of the disclosure can be stored atvarious predetermined temperatures and relative humidities for definedor predetermined time periods, for example in an open or closedcontainer. In some embodiments, formulations of the disclosure arestable upon storage at about 5, 25, 30, 37, 40 or 45 degrees Celsius andabout 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% relative humidity for a periodof at least about 0.5, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5,15, 20, 25, 30, 35, 40, 45, 48, 50, 51, 52, 53, 55 or 60 hours 1 week, 2weeks, 3 weeks or 4 week; 1 month, 2 months, 3 months, 4 months, 5months or 6 months.

In certain embodiments, formulations of the disclosure are stable uponstorage in an open or closed container at: about 30 degrees Celsius andabout 90 percent relative humidity for a period of at least about 20hours; about 40 degrees Celsius and about 60 percent relative humidityfor a period of at least about one week, two weeks or three weeks; about40 degrees Celsius and about 75 percent relative humidity for a periodof at least about one week, two weeks or three weeks; about 25 degreesCelsius and about 60 percent relative humidity for a period of at leastabout one month; about 40 degrees Celsius and about 75 percent relativehumidity for a period of at least one month; about 25 degrees Celsiusand about 75 percent relative humidity for a period of at least about 3months; or 5 degrees Celsius at any relative humidity for a period of atleast about three months. In some embodiments, “storage in an opencontainer” means that the container was opened twice a day for a givenperiod of time, for example up to four weeks, but was otherwise leftclosed.

In another embodiment, the formulation comprises the active ingredient2-(1-(4-((4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrileor the active ingredient2-(1-(4-((4-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrileand is stable upon storage in an open or closed container at: about 30degrees Celsius and about 90 percent relative humidity for a period ofat least about 20 hours; about 40 degrees Celsius and about 60 percentrelative humidity for a period of at least about one week, two weeks orthree weeks; about 40 degrees Celsius and about 75 percent relativehumidity for a period of at least about one week, two weeks or threeweeks; about 25 degrees Celsius and about 60 percent relative humidityfor a period of at least about one month; about 40 degrees Celsius andabout 75 percent relative humidity for a period of at least one month;about 25 degrees Celsius and about 75 percent relative humidity for aperiod of at least about 3 months; or 5 degrees Celsius at any relativehumidity for a period of at least about three months.

The pharmaceutical formulations of the disclosure can also be tested forother physical characteristics, for example by evaluating the amount ofactive ingredient and/or impurity levels of the formulations at the endof a predetermined time period after they have been subjected topredetermined conditions, for example, temperature and relative humidityin open and closed containers. Suitable methods for measuring theimpurity profile of the present formulations are known in the art.Exemplary methods for measuring the impurity profile of the presentformulations may involve any suitable chromatographic separationmethods, such as high-pressure liquid chromatography (HPLC) comprisingthe use of separation column and gradient elution as are known to thoseof ordinary skill in the art. An exemplary HPLC method for evaluatingthe amount of active ingredient and/or impurity levels of thepharmaceutical formulations of the present disclosure is described inExample 1 below. In addition, other methods may be utilized instead ofor in addition to HPLC separation methods, including capillaryelectrophoresis, electron paramagnetic resonance, gas-liquidchromatography, gravimetric analysis, solid-phase extraction methods,liquid-liquid extraction method, ultraviolet spectrometry, infraredspectroscopy, supercritical fluid extraction column chromatography, massspectrometry, nuclear magnetic resonance (NMR) spectroscopy, and RAMANspectroscopy.

In some embodiments, the impurity test comprises subjecting theformulation to storage conditions at 40 degrees Celsius at 75% relativehumidity in open and closed containers. In another embodiment, theimpurity test comprises subjecting the formulation to storage conditionsunder accelerated stress conditions at 60 degrees Celsius in closedcontainers. In another embodiment, the formulations may be evaluated forimpurity levels following storage at 40 degrees Celsius at 75% relativehumidity in open containers for one week.

The pharmaceutical formulations of the disclosure can also be tested forphysical characteristics, such as Vitamin E content, by use of anysuitable analytical method, for example an HPLC method comprisingisocratic elution with water and acetonitrile as the mobile phase and UVdetection as described in Example 1 below.

Although exemplary amounts or ranges for the active ingredient and othercomponents are given, pharmaceutical formulations of the disclosure cancomprise any amount of these components suitable for the purposes ofobtaining the desirable pharmacologic and stability properties asdescribed herein. In addition to the active ingredient, pharmaceuticalcompositions of the disclosure can also comprise other pharmaceuticallyacceptable excipients, for example adjuvants, antioxidants, binders,buffers, coatings, coloring agents, compression aids, diluents,disintegrants, emulsifiers, emollients, encapsulating materials,fillers, flavoring agents, glidants, granulating agents, lubricants,metal chelators, osmo-regulators, pH adjustors, preservatives,solubilizers, sorbents, stabilizers, sweeteners, surfactants, suspendingagents, thickening agents, or viscosity regulators. Suitable excipientsfor use in pharmaceutical compositions of the disclosure are described,for example, in the “Handbook of Pharmaceutical Excipients”, 5thEdition, Eds.: Rowe, Sheskey, and Owen, APhA Publications (Washington,D.C.), Dec. 14, 2005, the disclosure of which is incorporated herein byreference.

In certain embodiments, pharmaceutical compositions of the disclosurecan be compacted into a unit dose form, e.g., tablet or caplet, or addedto unit dose form, e.g., a compressed tablet. In a further embodiment,pharmaceutical compositions of the disclosure can be formulated foradministration as micronized granules or as a suspension of micronizedgranules. A pharmaceutical formulation of the disclosure which comprisesmicronized granules or a suspension thereof can, for example, besprinkled on or mixed with a semi-solid carrier such as apple sauce oranother food item for administration to a subject. The which comprisesmicronized granules or a suspension thereof can also, for example, beadded to a liquid carrier suitable for administration to subjects, suchas a solution of about 2% w/V hydroxypropyl cellulose and about 0.1% w/Vpolysorbate 80 in water or about 0.2% hydroxypropylcellulose, and 0.1%Tween 80 in water, to form a suspension.

In one embodiment, the dosage form of the disclosure comprises acompressed tablet, for example at about 25, 50, 75, 80 or 100 mgstrengths. In another embodiment, the dosage form of the disclosurecomprises a capsule, for example at about 25, 50, 75, 80 or 100 mgstrengths. In a further embodiment, the dosage form of the disclosure isa tablet comprising micronized granules of the active ingredient, forexample at about 25, 50, 75, 80 or 100 mg strengths. In anotherembodiment, the dosage form of the disclosure is a capsule comprisingmicronized granules for example at about 25, 50, 75, 80 or 100 mgstrengths. In one embodiment, the dosage form of the disclosure is acapsule comprising micronized granules for example at about 75 mgstrength.

Suitable techniques for formulating pharmaceutical compositions of thedisclosure into tablets are well-known in the art, and can comprisemixing the active ingredient and stabilizing components with one or morepharmaceutically acceptable tableting excipients and compressing themixture into a tablet, for example with a tableting press. The amountand nature of the tableting excipients used can be readily chosen basedon the desired characteristics of the tablet, such as size, hardness,friability and the like. Tablets comprising pharmaceutical compositionsof the disclosure can also be coated, for example with film coatingslike Opadry White®, or with enteric coatings designed to preventdissolution of the tablets until the transit the stomach and/or upperintestine. Suitable tablet coatings and methods for applying them arewell-known in the art.

Suitable techniques for formulating pharmaceutical compositions of thedisclosure into capsules are also well-known in the art, and cancomprise mixing the active ingredient and stabilizing components withone or more pharmaceutically acceptable capsule excipients and fillingthe mixture into a capsule. In one embodiment, a pharmaceuticalformulation of the disclosure (with or without additional excipients)can be filled into a capsule, such as a hard gelatin capsule. The hardgelatin capsule can be of any appropriate size, for example size ‘0’,‘0EL’, ‘3’, ‘4’ and the like. For example, in one embodiment a capsuleof the disclosure having a dosage strength of 25 mg of the activeingredient can be filled into a hard gelatin capsule of size 4, wherethe target capsule fill weight can comprise 100 mg. In anotherembodiment, a capsule of the disclosure having a dosage strength of 100mg of the active ingredient can be filled into a hard gelatin capsule ofsize 0 el, where the target capsule fill weight can comprise 400 mg.

Also provided herein are kits comprising at least one dosage form of thedisclosure, for example a tablet or capsule, and instructions foradministering the at least one dosage form to a subject. The kit canalso comprise packaging or a container housing the at least one dosageform of the disclosure, and can also comprise instructions on storage,administration, dosing or the like and/or an insert regarding the activeingredient. The kit can also comprise instructions for monitoringcirculating levels of the active ingredient (or metabolites thereof)once administered, and optionally materials for performing such assaysincluding, e.g., reagents, well plates, containers, markers or labels,and the like. Other suitable components to include in kits of thedisclosure will be readily apparent to one of skill in the art, takinginto consideration the desired indication, dosing regimen, storageconditions and route of administration.

The pharmaceutical compositions of the disclosure can formulated foradministration as a single dose or as multiple doses for continuous orperiodic discontinuous administration. For continuous administration, akit can include the pharmaceutical compositions of the disclosure inindividual unit dosage forms (e.g., tablet or capsule), and optionallyinstructions for administering the individual unit dosage forms, forexample, more than once daily, twice a day (BID), four times a day(QID), daily, weekly, or monthly, for a predetermined length of time oras prescribed. When the pharmaceutical compositions of the disclosureare to be delivered periodically in a discontinuous fashion, a kit caninclude placebos during periods when the individual unit dosage formsare not delivered. In some embodiments, formulations of the presentdisclosure can be administered at a dose of about 10 mg BID, about 20 mgBID, about 30 mg BID, about 40 mg BID, about 50 mg BID, about 75 mg BID,about 100 mg BID, about 80 mg QID, or about 120 mg QID. In oneembodiment, formulations of the present disclosure can be administeredat a dose of about 75 mg BID.

Suitable packages or containers are known in the art for holding anddispensing pharmaceutical agents for periodic oral use. In oneembodiment, the package comprises indicators for each administrationperiod. In another embodiment, the package comprises a labeled blisterpackage, dial dispenser package, or bottle. The kits of the disclosurecan also comprise a means for containing any type of packaging thathouses the unit dosage forms, for example bottles or vials, which can(for example) be held in close confinement for commercial sale such as,e.g., injection or blow-molded plastic containers into which the bottlesor vials are retained.

The pharmaceutical compositions, methods of treatment, dosage forms andkits of the disclosure are useful in treating conditions which areassociated with dysregulation (e.g., abnormality or impairment) of theSyk/JAK pathway. In one embodiment, the conditions are associated withdysregulation of the Syk/JAK2 pathway. In one embodiment, a conditionassociated with dysregulation of the Syk/JAK pathway comprises acute orchronic inflammatory disorders, such as atopic dermatitis. In oneembodiment, a condition associated with dysregulation of the Syk/JAKpathway comprises a disease that is associated with abnormal cellularproliferation. The term “abnormal cellular proliferation” refers to theuncontrolled growth of cells which are naturally present in a mammalianbody. In one embodiment, a disease which is characterized by abnormalcellular proliferation is cancer, for example cancer of the prostate,head, neck, eye, mouth, throat, esophagus, bronchus, larynx, pharynx,chest, bone, lung, colon, rectum, stomach, bladder, uterus, cervix,breast, ovaries, vagina, testicles, skin, thyroid, blood, lymph nodes,kidney, liver, intestines, pancreas, brain, central nervous system,adrenal gland, skin or a leukemia or lymphoma. In one embodiment, thedisease characterized by abnormal cellular proliferation is cancer ofthe prostate. In another embodiment, the abnormal cellular proliferationis associated with at least one solid tumor.

In one embodiment, the pharmaceutical compositions, methods oftreatment, dosage forms and kits of the disclosure are useful intreating conditions including (without limitation) peripheral T-celllymphoma (PTCL), chronic lymphocytic leukemia (CLL), myelofibrosis (MF),for example primary myelofibrosis (PMF), essential thrombocytopenia(ET), and polycythemia vera (PV), mature B-cell neoplasms, for examplediffuse large B-cell lymphoma (DLBCL), both germinal B-cell (GCB) asactivated B-cell (ABC) subtypes, mantle cell lymphoma, high grade B-celllymphoma (HGBL), anaplastic large cell lymphoma, marginal zone lymphoma,hairy cell leukemia, Waldenstrom macroglobulinemia, Monoclonalgammopathy of undetermined significance (MGUS), plasma cell myeloma,Burkitt lymphoma, Mature T and NK neoplasms, Hodgkin lymphoma,posttransplant lymphoproliferative disorders (PTLD), histiocytic anddendritic cell neoplasms, myeloid neoplasms, for example PV, ET, primarymyelofibrosis, chronic neutrophilic leukemia, chronic myeloid leukemia,atypical chronic myeloid leukemia, juvenile myelomonocytic leukemia, andacute myeloid leukemia, precursor lymphoid neoplasm, for example, B-cellacute lymphoblastic leukemia (B-ALL), Down Syndrome ALL, T-cell ALL(T-ALL), Mature lymphoid neoplasms, for example, T-cell prolymphocyticleukemia, adult T-cell leukemia/lymphoma (ATLL), Natural Killer/T-celllymphoma (NK/TCL), NK/T—Large Granular Lymphocytic Leukemia (NK/T-LGL),primary mediastinal large B-cell lymphoma/Hodgkin lymphoma (PBMCL/HL)and follicular lymphoma (FL), primary cutaneous lymphoma (PCL), forexample, mycosis fungoides/Sezary syndrome, and/or peripheral T-celllymphoma (PTCL). Further conditions include (without limitation) acuteand chronic graft-versus host Disease (aGVHD and cGVHD) and treatment ofimmune mediated complications of checkpoint inhibitors or otherimmune-oncology therapies. A list of JAK or SYK driven hematologicmalignancies is found in the 2016 revision of the World HealthOrganization (WHO) classification of lymphoid neoplasms; Blood 2016127:2375-2390, which is incorporated by reference herein. A list ofhemotologic malignancies with known JAK mutations is found atHaematologica. 2015 October; 100(10): 1240-1253, which is incorporatedby reference herein.

In another embodiment, a condition associated with dysregulation of theSyk/JAK pathway comprises acute or chronic inflammatory disorders, suchas neutrophil-associated inflammation, inflammatory arthritis,inflammation in peritonitis, inflammation after myocardial infarction orbleomycin-induced pulmonary fibrosis. Models for testing the ability ofcompounds to reduce inflammation in inflammatory arthritis are known,e.g., as described by Camps et al, Nature Med., 2005, 11, 936-943, whichalso describes models useful in assessing the ability of compounds toreduce inflammation in peritonitis; models for testing the ability ofcompounds to reduce inflammation and/or improve healing after myocardialinfarction are described by Siragusa et al, Circ. Res. (2010), 106,757-768; and a model for testing the ability of compounds to preventbleomycin-induced pulmonary fibrosis is described by Wei et al, BiochemBiophys Res Comm. 2010, 397: 311-317 and Brent et al, Toxicology, 2000,147: 1-13, the entire disclosures of which are incorporated herein byreference.

In one embodiment, the present disclosure provides formulations,methods, kits, and dosage forms for broadly treating all autoimmunediseases, including, for example (without limitation), atopicdermatitis, alopecia areata, hand and foot eczema, hidradenitissuppurativa, pemphigus vulgaris, psoriasis, cutaneous lupus, vitiligo,inflammatory bowel disease (UC, CD), rheumatoid arthritis, asthma,allergic rhinitis, systemic lupus erythematosus (SLE), psoriaticarthritis, and multiple sclerosis (including other autoimmune diseases).

The disclosure thus provides a method of treating a diseasecharacterized by the dysregulation (e.g., abnormality or impairment) ofthe Syk/JAK pathway in a subject, comprising administering to thesubject a therapeutically effective amount of an active ingredient inone or more dosage forms, wherein the dosage forms comprise apharmaceutical formulation comprising an active ingredient, wherein theactive ingredient in the form of micronized granules is formed into acompressed tablet, wherein the active ingredient comprises a compound ofthe formula (I), and wherein the active ingredient retains stabilityafter storage of the pharmaceutical formulation for a predetermined timeand under predetermined conditions.

As described herein, a therapeutically effective amount of an activeingredient of the disclosure when used for the treatment of cancer is,for example, an amount which may reduce the number of cancer cells influids (e.g., blood, peripheral cells or lymphatic fluids), reduce tumorsize, inhibit metastasis, inhibit tumor growth and/or ameliorate one ormore of the symptoms of the cancer. For cancer therapy, efficacy can bemeasured for example, by assessing the time to disease progressionand/or determining the response rate, or measuring inhibition of tumorgrowth or metastasis. In one embodiment, administration of theformulations described herein can achieve inhibition of tumor growth inan amount of 0% to 100%, preferably an amount of above about 50%.

As described herein, a therapeutically effective amount of an activeingredient of the disclosure when used for the treatment of aninflammatory disorder, such as atopic dermatitis, is an amount which maydelay the onset of or reduce the severity or duration of an inflammatoryresponse, or which mitigates one or more symptoms of an inflammatoryresponse. For treatment of an inflammatory disorder, efficacy can bemeasured, for example, by a reduction in physiologic signs ofinflammation (e.g., redness, swelling, heat, loss of function) or bymeasuring changes in the levels of cells (e.g., monocytes, macrophagesand other mononuclear cells) or molecules (e.g., pro-inflammatorycytokines) associated with inflammation. In one embodiment, treatment ofatopic dermatitis can be measured by evaluating a subject according tothe Investigators Global Assessment (IGA) scale, the 5-D Pruritus Scale,the Pruritus Numeric Rating Scale or the Eczema Area and Severity Index(EASI) assessment tool, as described, for example, in FIGS. 3 and 4 andin Example 3 below.

The Syk/JAK pathways are known to be deregulated in various cancers dueto specific mutations in different members of each pathway. For exampleaberrations in Syk/JAK pathways, such as those caused by the recentlyidentified JAK2^(V617F) mutation and translocations of the JAK2 gene,are underlying causes of leukemias and other myeloproliferativedisorders. Such mutations are easily detected in tumor samples usingmethods known in the art Sarkar et al (Diagn Mol Pathol. (1995)4(4):266-73), the entire disclosure of which is herein incorporated byreference.

Identifying a mammalian subject, e.g., a human patient, or a populationof such subjects who will respond positively to treatment withpharmaceutical formulations of the disclosure prior to initiation oftreatment (also termed herein “predetermining” or “selecting”) can beaccomplished by assaying a sample (for example a tumor biopsy or bloodsample comprising white blood cells when the condition is cancer) from apatient to detect one or more of the Syk/JAK mutations discussed above.Upon detection of a Syk/JAK mutation, the subject may be treated withthe pharmaceutical formulations of the present disclosure, for exampleby administering one or more pharmaceutical formulations of the presentdisclosure which comprise a therapeutically effective amount of anactive ingredient as described herein.

A suitable sample may be obtained from the body of a subject and mayinclude, e.g., tissue samples, cells, extracellular matter, orcirculating cancer cells in blood or lymphatic fluid. Tissue samples maybe from any organ, including disease states of such organs, such as theskin, the blood circulatory system, and any circulating tumor cells.Tissue samples such as tumor biopsies may be obtained using knownprocedures. Tissue specimens may also include xenograft tumor samples,e.g., those from animals used in drug dose or toxicology studies.

For example, a subject can be tested for the presence of a JAK2^(V617F)mutation. As discussed above, these mutations can be detected using anysuitable technique known in the art, including fluorescence in situhybridization, PCR-based sequencing of relevant portions of a givengene, restriction fragment length polymorphism analysis, or bymonitoring expression levels of a given gene product (e.g., protein orRNA. In one embodiment, a method is provided for treating a conditiontreatable by inhibiting the Syk/JAK pathway, comprising selecting asubject who has a JAK2^(V617F) mutation; and administering atherapeutically effective amount of a pharmaceutical formulation of thedisclosure. In one embodiment, a method is provided for treatingpatients whose cancers are characterized by the presence of theJAK2^(v617F) mutation and translocation of the JAK2 gene comprising thesteps of identifying patients having such mutation(s) and administeringa therapeutically effective amount of the formulation disclosed herein.

In an embodiment, the present disclosure provides pharmaceuticalformulations comprising granules, wherein the granules comprise:micronized active ingredient; one or more granulation binders; one ormore fillers; one or more disintegrants; and one or more antioxidants,wherein the active ingredient is a compound of Formula (I) or apharmaceutically acceptable salt or prodrug thereof, and wherein theformulation may further comprise extragranular components. In anembodiment, the active ingredient comprises2-(1-(4-((4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile.In another embodiment, the active ingredient comprises2-(1-(4-((4-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile.

In an embodiment, the antioxidants of the formulations described hereinmay include vitamin E or butylated hydroxytoluene; the fillers maycomprise lactose monohydrate; the distintegrants may comprisecrospovidone or croscarmellose sodium and the granulation binders maycomprise polyvinylpyrollidone or hydroxypropylcellulose. In certainembodiments, the granulation binders comprise hydroxypropylcellulosehaving a viscosity at 25° C. of 75-150 centipoise in a 5% w/w in aqueoussolution. In certain embodiments, the granulation binders comprisepolyvinylpyrollidone having a number average molecular weight of about30,000. In an embodiment, the micronized granules of the presentlydescribed formulation have a particle size of less than about 20microns. In an embodiment, the micronized granules have an isopropylalcohol content of less than about 5000 ppm.

In certain embodiments, the formulations may comprise one or moreextragranular components. The extragranular components may comprise oneor more tableting fillers, one or more disintegrants, one or morelubricants, and optionally one or more surfactants. In certainembodiments, the tableting fillers may comprise microcrystallinecellulose, the disintegrants may comprise croscarmellose sodium, thesurfactants may comprise sodium lauryl sulfate, and the lubricants maycomprise magnesium stearate. In an embodiment, the micronized granulesand extragranular components may be compressed into a tablet.

The formulations of the present disclosure may exist in any embodimentknown to one skilled in the art. In an embodiment, the formulation maybe present in the form of tablets, scored tablets, compressed tablets,coated tablets, capsules, caplets, pills, powder packets andmodifications thereof. In an embodiment, the formulation describedherein comprises compressed tablets.

In an embodiment the micronized granules of the present formulation havean isopropyl alcohol content of less than about 5000 ppm. The activeingredient in the embodiments described herein may be between about 5 to50 mg, 5 mg, about 20 mg, or about 50 mg. Other aspects of theembodiments described herein may include a tablet hardness ofapproximately 5-12 kP, or 7 to 9 kP and a disintegration time of lessthan about 5 minutes in 0.1 N HCl, pH 6.8 and 50 mM phosphate buffer at37° C. In certain embodiments the formulations may comprise a tablethaving an aesthetic coating; the coating may be comprised ofhydroxypropylcellulose, titanium dioxide, talc and polyethylene glycol.

In an embodiment, the formulation described herein comprises acompressed tablet having micronized granules and extragranularcomponents, wherein: the tablet has a total weight of about 150 mg; themicronized granules comprise about 5 to 50 mg of an active ingredient,about 75 to 900 mg, or 118.6 to 736 mg of lactose monohydrate, about1-20 mg, or 4.5 mg croscarmellose sodium, about 0.1-5 mg or 0.15 mgvitamin E, and about 1-10 mg, or 4.5 mg granulation binder; furthermore,the extragranular components may comprise about 5-20 mg, or 11.25 mg ormicrocrystalline cellulose, about 1-10 mg or 4.5 mg croscarmellosesodium, about 1-10 mg or 1.5 mg magnesium stearate, and about 1-10 mg or6 mg of an enteric coating. The active ingredient in such an embodimentmay comprise2-(1-(4-((4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile.

In certain embodiments of such a compressed tablet, granulation bindersmaybe selected from the group consisting of polyvinylpyrollidone andhydroxypropylcellulose wherein hydroxypropylcellulose has a viscosity at25° C. of 75-150 centipoise in a 5% w/w in aqueous solution. In certainembodiments, the micronized granules comprise about 75-150 mg, or 117.1mg to about 72.1 mg of lactose monohydrate and the extragranularcomponents further comprise about 1-10 mg or 1.5 mg sodium laurylsulfate.

In certain embodiments, the micronized granules of a compressed tabletas described herein may comprise about 5 mg of an active ingredient andabout 118.6 mg of lactose monohydrate, about 20 mg of an activeingredient and about 103.6 mg of lactose monohydrate, or about 50 mg ofan active ingredient and about 73.6 mg of lactose monohydrate.

Further included herein are embodiments comprising methods ofmanufacturing the pharmaceutical formulation embodiments describedabove. Also included are methods of preparing compressed tablets andmethods of stabilizing pharmaceutical formulations as well as thepreparation of dosage forms comprising micronized granules andextragranular components compressed into a tablet. In an embodiment, themethods, protocols and procedures regarding the foregoing comprise theincorporation of an active ingredient together with antioxidantsincluding vitamin E or butylated hydroxytoluene; fillers comprisinglactose monohydrate; distintegrants comprising crospovidone orcroscarmellose sodium and granulation binders comprisingpolyvinylpyrollidone or hydroxypropylcellulose. In certain embodiments,the granulation binders comprise hydroxypropylcellulose having aviscosity at 25° C. of 75-150 centipoise in a 5% w/w in aqueoussolution. In certain embodiments, the granulation binders comprisepolyvinylpyrollidone having a number average molecular weight of about30,000. In an embodiment, the micronized granules of the presentlydescribed embodiments have a particle size of less than about 20microns. In an embodiment, the micronized granules have an isopropylalcohol content of less than about 5000 ppm.

In an embodiment, the formulations may further comprise one or moreextragranular components. The extragranular components may comprise oneor more tableting fillers, one or more disintegrants, one or morelubricants, and optionally one or more surfactants. In certainembodiments, the tableting fillers may comprise microcrystallinecellulose, the disintegrants may comprise croscarmellose sodium, thesurfactants may comprise sodium lauryl sulfate, and the lubricants maycomprise magnesium stearate. In an embodiment, the micronized granulesand extragranular components may be compressed into a tablet.

In an embodiment, the active ingredient for the foregoing may comprise2-(1-(4-((4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile.In another embodiment, the active ingredient may comprise2-(1-(4-((4-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile.

In the above embodiments, the methods of manufacturing and producing maycomprise mixing intragranular ingredients comprising an activeingredient; one or more fillers; one or more disintegrants; and one ormore antioxidants; granulating the mixed intragranular ingredients whileadding a solution of 10% w/w of one or more granulation binders in 99%v/v isopropyl alcohol until granules are formed; drying and milling thegranules to make micronized granules; wherein the active ingredientcomprises a compound of Formula (I) or a pharmaceutically acceptablesalt or prodrug thereof.

In a further embodiment, kits comprising one or more dosage forms andinstructions for administering the dosage forms to a subject, whereinthe dosage forms comprise granules and extragranular componentscompressed into a tablet, are also provided. In such embodiments, theformulation may comprise the active ingredients comprising2-(1-(4-((4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile.In another embodiment, the active ingredient may comprise2-(1-(4-((4-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile.

The above embodiments may be utilized in methods for treating cancer oratopic dermatitis or inflammation in a subject, comprising administeringto the subject a therapeutically effective amount of an activeingredient in one or more dosage forms, wherein the dosage formscomprise micronized granules and extragranular components compressedinto a tablet.

Active ingredients of the present disclosure can be prepared, forexample, according to the methods disclosed in U.S. Pat. Nos. 8,729,079and 9,382,277, the entire disclosures of which are herein incorporatedby reference. In some embodiments of the disclosure, an activeingredient comprising the pharmaceutical formulation of the disclosurecan be present in at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% w/w.

The active ingredient for use in the present formulations and methodscomprises compounds which regulate the Syk/JAK pathway. The regulatoryactivity of the active ingredients of the disclosure makes thesecompounds useful for manufacturing pharmaceutical formulations, whichcan be used for treating conditions such as inflammatory disorders,including atopic dermatitis, or cancers characterized by the presence ofsolid tumors, particularly melanoma, colon cancer, non-small cell lungcancer, bladder cancer, and breast cancer.

The therapeutically effective amount of a pharmaceutical formulation ofthe disclosure provided to a subject will vary depending upon thepurpose of the administration, the state of the patient, level ofdisease penetration and the like. As used herein, “subject” includes anyhuman or non-human animal in need of treatment with the pharmaceuticalformulations of the disclosure. In one embodiment, a subject is anyhuman in need of treatment with the formulations of the disclosure(sometimes referred to herein as a “patient”). A therapeuticallyeffective amount of the active ingredient in the pharmaceuticalformulations of the disclosure can be determined by an ordinarilyskilled physician or medical professional, taking into account certainvariables, including the specific condition and the size, age, weight,gender, disease penetration, previous treatment and response pattern ofthe patient.

In one embodiment, the pharmaceutical formulation is administeredorally, for example in capsule or tablet form. For example, the presentformulations can be provided as a unit dose, for example as a compressedtablet, comprising a therapeutically effective amount. In oneembodiment, a unit dose comprising the pharmaceutical formulation of thedisclosure can be administered once daily or multiple times daily, forexample, 1 to 6 times in a 12 or 24 hour period. If multiple unit dosesare administered in a given time period, they can be administered atsubstantially even time intervals. For example, if two unit doses areadministered in a 12 hour period, they can be given to the patient 6hours apart. Multiple unit doses are administered in a given time periodcan also be administered at substantially uneven time intervals. In oneembodiment, a unit dose comprises a dosage form of the disclosure in theform of a tablet or capsule for oral administration.

In some embodiments, the active ingredient in the pharmaceuticalformulations of the disclosure can comprise an amount of about 0.5 to100 percent by weight, for example about 0.5, 1, 1.5, 2, 2.5, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99 or 100 percent by weight. In another embodiment, the activeingredient comprises about 3.5 percent or about 14 percent of thepharmaceutical formulation by weight.

In some embodiments, formulations of the disclosure comprise an activeingredient of the disclosure, formed into oral dosage forms such astablets, capsules, powders, suspensions, and the like. In such dosageforms of the disclosure, the amount of active ingredient comprising thedosage form can be any suitable amount, for example about 0.5, 1, 1.5,2, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 96, 97, 98, 99 or 100 mg per unit dosage form. In certainembodiments, dosage forms of the disclosure comprise about 20 to about80 mg of the active ingredient per dosage form.

In certain embodiments, formulations of the disclosure comprise anactive ingredient of the disclosure, formed into dosage forms such astablets, capsules, sachets, powders, suspensions, suppositories and thelike. In such dosage forms of the disclosure, the amount of activeingredient comprising the dosage form can be any suitable amount, forexample about 0.5, 1, 1.5, 2, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100 mg perunit dosage form. In certain embodiments, dosage forms of the disclosurecomprise about 25, 50, 75, 80 or 100 mg of the active ingredient perdosage form. In one embodiment, dosage forms of the disclosure compriseabout 75 mg of the active ingredient per dosage form.

A suitable daily (i.e. 24 hour time period) dose according to methods ofthe disclosure, whether given all at once or in multipleadministrations, can depend on the specific method of treatment andcondition treated. In one embodiment, a suitable daily dose, whethergiven all at once or in multiple administrations, is between about 10 to120 mg for oral application, for example about 20 mg to 80 mg, 25 to 75mg, 30 mg to 70 mg, 35 mg to 65 mg, or 40 mg to 60 mg. In oneembodiment, a suitable daily dose is between about 40 mg to about 80 mg.In other embodiments, a suitable daily dose is about 10 mg, 15 mg, 20mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg or 120 mg. In oneembodiment, a suitable daily dose is about 20 mg. In another embodiment,a suitable daily dose is about 40 mg. In another embodiment, a suitabledaily dose is about 80 mg.

In another embodiment, a suitable daily (i.e. 24 hour time period) doseaccording to methods of the disclosure, whether given all at once or inmultiple administrations, can depend on the specific method of treatmentand condition treated. In one embodiment, a suitable daily dose, whethergiven all at once or in multiple administrations, is between about 10 to1000 mg for oral application, for example about 20 to 500 mg, 50 mg to250 mg or 75 mg to 100 mg. In other embodiments, a suitable daily doseis about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg,55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg,200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mgor 1000 mg.

In another embodiment, a suitable daily dose, whether given all at onceor in multiple administrations, is about 0.1 mg/kg to about 100 mg/kg,about 0.5 mg/kg to about 75 mg/kg, about 0.1 mg/kg, 1 mg/kg, 10 mg/kg,20 mg/kg, 30 mg/kg, 40 mg/kg, 50 μg/kg, 75 μg/kg or 100 μg/kg.

The therapeutically effective amounts may be provided on regularschedule, i.e., daily, weekly, monthly, or yearly basis or on anirregular schedule with varying administration days, weeks, months, etc.Alternatively, the therapeutically effective amount to be administeredmay vary. In one embodiment, the therapeutically effective amount forthe first dose is higher than the therapeutically effective amount forone or more of the subsequent doses. In another embodiment, thetherapeutically effective amount for the first dose is lower than thetherapeutically effective amount for one or more of the subsequentdoses. Equivalent dosages may be administered over various time periodsincluding, but not limited to, about every 2 hours, about every 6 hours,about every 8 hours, about every 12 hours, about every 24 hours, aboutevery 36 hours, about every 48 hours, about every 72 hours, about everyweek, about every two weeks, about every three weeks, about every month,and about every two months. Alternatively, equivalent doses may beadministered over uneven intervals in accordance with the recommendedtreatment of a health-care practitioner. The number and frequency ofdosages corresponding to a completed course of therapy will bedetermined according to the judgment of a health-care practitioner. Thetherapeutically effective amounts described herein refer to totalamounts administered for a given time period; that is, if more than oneactive ingredient is administered, the therapeutically effective amountscorrespond to the total amount administered.

In one embodiment, the pharmaceutical formulation is administered orallyonce a day (QD). The administration can be short-term or long-term. Forexample, short term administration can comprise administration of thepharmaceutical formulation once a day for about 1 week, 2 weeks, 3 weeksor 4 weeks, or any other longer or shorter term. For example, long termadministration can comprise administration of the pharmaceuticalformulation once a day for at least 1 week, 2 weeks, 3 weeks, 4 weeks,30 days, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, or 5years, or any other longer or shorter term.

The following examples are given to illustrate exemplary embodiments ofthe present disclosure. It should be understood, however, that thepresent disclosure is not to be limited to the specific conditions ordetails described in these examples.

EXAMPLES Example 1 Preformulation, Formulation and Analytical Data

Analytical studies for2-(1-(4-((4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrileare provided below in Tables 1-3: Table 1 provides the results of an HClpH dependent solubility profile study, Table 2 provides the results ofan HCl solubility profile in organic solvents, and Table 3 provides theresults of an HCl stress study.

TABLE 1 pH 4.5 pH 5.5 pH 6.8 pH 7.5 0.1N Acetate Acetate PhosphatePhosphate HCl Buffer Buffer Buffer Buffer Water Initial N/A 4.50 5.526.80 7.55 N/A pH End pH 1.23 4.56 5.12 6.86 6.96 2.62 Solu- 10810 2.91.0 2.7 0.7 941 bility μg/mL

TABLE 2 MeOH EtOH IPA Solubility μg/mL 4437 4996 462

TABLE 3 1N 1N HCl NaOH 3% H2O2 rt, rt, rt, 80° C. UV 24 hr 24 hr 30 hr24 hr 23 hr % API based on 100.00% 99.54% 83.00% 99.93% 99.80% Peak Area%

Studies were conducted to evaluate excipient compatibility, stabilityand disintegration comprising the use of Ac-Di-Sol and PolyplasdoneXL-10; PVP K-30 and Klucel LF and SLS. Water was avoided as agranulating solved and IPA was used. The coating comprised Opadry(IPA/water, 50/50) and tables were prepared in 5/20/50 mg strength, 150mg weight (DT:<5 min in 0.1 N HCl, pH 6.8, 50 mM PO4 buffer). Detailsconcerning ingredients and relative weights for prototype formulation Aand formulation B are provided below in Table 4.

TABLE 4 Formulation A Batch Formulation B Batch Ingredients mg/tab (g)mg/tab (g) Intragranular API 5/20/50 1.50 5 1.50 Lactose Monohydrate117.25/lower 35.18 118.75 35.63 Primellose 4.5 1.35 0 0.00 PolyplasdoneXL-10 0 0.00 4.5 1.35 Klucel LF/PVP K-30 4.5 1.35 4.5 1.35 ExtragranularVivapur 101 11.25 3.38 11.25 3.38 Primellose 4.5 1.35 0 0.00Polyplasdone XL-10 0 0.00 4.5 1.35 Magnesium Stearate 1.5 0.45 1.5 0.45SLS 1.5 0.45 0 0.00 Total weight 150 45 150 45.00 Coating: 4% w/w,Opadry White

In an additional study, residual solvent was evaluated in the tableswherein IPA was used as a granulation solvent. The results are providedbelow in Table 5.

TABLE 5 Sample Reported Sample ID Weight (mg) Content (ppm) P14K080002A108.84 387 P14K080002B 107.48 292 P14K080003A 100.35 1272 P14K080003B102.15 470 P14K080004A 102.65 643 P14K080004B 102.90 164

The formulation screening strategy comprised a first batch screeningunder accelerated storage conditions at 40 C/75% RH in open and closedcontainers. Another batch was screened under accelerated stressconditions at 60 C in closed containers. Stability data is provided inTable 6.

TABLE 6 1 Week 40° C./75% Initial RH Open 1 Week 60° C. Sample TotalTotal Total Information Composition Assay, % Impurity, % Assay, %Impurity, % Assay, % Impurity, % P14K080001A PVP/AC-Di-Sol/SLS 98.60.436 98.9 0.602 90.6 2.157 P14K080001B PVP/Polyplasdone XL 101.4 0.33494.1 2.673 101.1 2.629 10 P14K080001C PVP/AC-Di-Sol 103.0 0.275 104.50.476 102.4 1.156 P14K080001D PVP/Polyplasdone XL 107.6 0.370 104.21.002 107.6 2.082 10/SLS P14K080002A KlucelLF/AC-Di- 106.9 0.222 107.80.210 105.0 1.653 Sol/SLS P14K080002B KlucelLF/ 104.6 0.364 104.4 0.143104.7 2.100 Polyplasdone XL10 P14K080002C KlucelLF/AC-Di-Sol 104.6 0.33096.7 2.480 105.8 0.259 P14K080002D Klucel LF/ 98.4 0.334 93.4 3.759 97.62.230 Polyplasdone XL 10/SLS

The analytical procedures for determination of content uniformity, blenduniformity, amount of active ingredient and related impurities(including degradation compounds) in pharmaceutical formulations of thepresent disclosure comprising active ingredient-containing tablets (5mg, 20 mg and 50 mg strengths) comprised the use of an HPLC method. Thecolumn used for separation has USP L1 packing and dimensions of 4.6×150mm, with a 3.5 micron particle size. The HPLC method uses gradientelution where the mobile phase was a buffer solution of 1 mM ammoniumformate, pH 3.2, and 0.1% formic acid in acetonitrile, and elutedfractions were subject to UV detection at 275 nm. Detailed analyticalresults for prototype formulations at 1 week, 40 C/75% RH in an opencontainer are provided in Table 7.

TABLE 7 1 WK, 40° C./75% RH, Open Batch # Wt of Tablets (g) ImpuritiesRetention Time, min RRT Peak Area % Recover P14K080002A 1.60316Unknown-1 7.105 0.67 1258 0.002 Unknown-2 7.399 0.70 2125 0.004Unknown-8 9.79 0.93 5716 0.011 Unknown-10 10.315 0.98 7262 0.014 ASN00210.559 1.00 40910752 N/A Unknown-12 11.303 1.07 29691 0.051 Unknown-1311.593 1.10 21378 0.043 Unknown-14 11.835 1.12 2951 0.005 Unknown-1512.187 1.15 4399 0.009 Unknown-16 12.428 1.18 1716 0.003 Unknown-1712.771 1.21 5924 0.012 Unknown-21 13.721 1.30 12305 0.025 Unknown-2514.568 1.38 2464 0.005 Unknown-26 14.86 1.41 1345 0.002 Unknown-2715.166 1.44 2264 0.004 Unknown-36 18.288 1.73 4677 0.009 % TotalImpurity 0.210 P14K080002B 1.58841 Unknown-2 7.388 0.70 2690 0.005Unknown-7 9.344 0.88 13003 0.026 Unknown-8 9.769 0.93 8669 0.017Unknown-9 10.085 0.96 2400 0.004 Unknown-10 10.294 0.97 14157 0.019ASN002 10.56 1.00 27284322 N/A Unknown-11 10.891 1.03 13904 0.049Unknown-12 11.302 1.07 402914 0.828 Unknown-13 11.595 1.10 18407 0.037Unknown-14 11.841 1.12 2100 0.004 Unknown-15 12.201 1.16 6897 0.014Unknown-16 12.432 1.18 3066 0.006 Unknown-17 12.798 1.21 10370 0.021Unknown-18 13.078 1.24 5544 0.011 Unknown-20 13.567 1.29 15836 0.032Unknown-25 14.628 1.39 6789 0.013 Unknown-27 15.171 1.44 2786 0.005Unknown-30 16.521 1.56 1719 0.003 Unknown-31 16.757 1.59 2345 0.004Unknown-35 18.071 1.71 5593 0.011 Unknown-36 18.259 1.73 9928 0.020Unknown-38 19.189 1.82 2262 0.004 % Total Impurity 1.143

The formulation batches described above were prepared by micronizing theactive, ingredient, combining the active ingredient with intragranularand extragranular components to form a tablet, and finally coating thetablet.

Micronization:

The general procedure for micronizing the active ingredient comprisedthe use of a Micronizer 4″ SDM and batches of approximately 500-550 gwere micronized. The micronization parameters consisted of thefollowing:

Inlet Air-100 PSI

Grinding Chamber Air-40 PSI

Feeder Nozzle-20 PSI

Number of Passes #1

In an embodiment, 512 g of material was added to the feeding chamber andthe weight of the material obtained after micronization was 452 g.

The table below provides particle size measurement in hexane for theactive ingredient as described herein.

TABLE 8A Active Ingredient D (0.1) D (0.5) D (0.9) As is API 3.75212.354 32.411 Micronized API 1.675 7.036 13.530

FIG. 1 provides a particle size distribution results analysis report forthe active ingredient prior to micronization, and FIG. 2 provides aparticle size distribution results analysis report for the activeingredient after micronization.

Formulation Configuration:

The formulations were configured into two tablet formulations, 20 mg,and 50 mg. Table 8b provides the evaluation of residual solvent levels(isopropyl alcohol or “IPA”) for the 20 mg tablet, and Table 8c providesPVP K30-IPA evaluation of IPA residual solvent levels for the 50 mgtablet. The general procedure for making the formulations comprisedweighing the required intragranular material and mixing it, tituratingthe powder mixer in a mortar and pestle, slowing adding IPA-PVP K30 10%w/w solution to the powder while continuingly mixing (adding additionalIPA 99% until granules are formed if required), drying the wet mass inan oven at 40 C for 30 minutes, passing the dried mass through Fitzmill(Hammer Forward, Screen #0040, Speed #2500 RPM), weighing and measuringthe required extragranular materials and adding them in following orderVivapur 101, Primellose/Polyplasone XL-10 and SLS, mixing for 3-5minutes in a tubular mixer, adding magnesium stearate and mixing for 1minute in a tubular mixer, tableting the formulation in F-Press keepingtarget at 150 mg and hardness at 9 KPa.

The coating formula is provided below in Table 8d:

TABLE 8d # Mg per Dose % w/w Ingredients Theoretical (g) 1 150 n/a APItablets 300 2  6 4 Opadry White 03F180004 12 3 n/a n/a Purified Water 544 n/a n/a Isopropyl Alcoholo 99% 54

Stability data was generated and collected for eight prototypeformulations. Details of the study results are proved below in Table 9a(40 degrees Celsius/75 RH) and Table 9b (60 degrees Celsius).Formulations 1A, 1C, 2A and 2C displayed superior stability results.

Formulations 1A, 1C, 2A and 2C were further evaluated to test the effectof antioxidants.

As shown in Tables 10a and 10b, certain prototype formulations wereevaluated for determination of DL-α-Tocopherol (Vitamin E) contentcomprising an HPLC method. The column used for separation has a USP L1packing, 4.6×150 mm, with a 3.5 micron particle size, and method usesisocratic elution with water and acetonitrile (97:3 v/v) as the mobilephase and UV detection at 294 nm.

Further analysis was conducted to evaluate excipient compatibility,stability and disintegration at the 5 mg strength: Ac-Di-Sol andPolyplasdone XL-10; PVP K-30 and Klucel LF; and SLS. In addition, higherstrength tablets were evaluated for stability and tablet properties, theeffect of antioxidants (5 mg strength), and polymorphs. Formulations A,B, C, and D provided in Table 11 below were selected for furtherevaluation.

Following additional analysis, the following formulations (Table 12)were selected as lead candidates:

Immediate-Release Formulations

Immediate-release formulations of the disclosure containing a micronizedhydrochloride salt of the active ingredient described herein wereprepared in 2 dosage strengths (5 mg and 20 mg) for use in clinicalstudies. Table 13 provides component composition and amounts for 5 mgand 20 mg strength tablets.

TABLE 13 Amount per Tablet (mg) Component 5 mg 20 mg Compound 1 HCl(micronized) 5.396** 21.584** Lactose Monohydrate, NF (Modified SprayDried 116.704 100.516 Fast Hydroxypropyl Cellulose, NF (Klucel ELF 4.5004.500 Pharm) Croscarmellose Sodium, NF (Ac-Di-Sol) 9.000 9.00 Vitamin E,USP (dl-α-Tocopherol) 0.150 0.150 Microcrystalline Cellulose, NF(Vivapur Type 11.250 11.250 101) Sodium lauryl sulfate, NF (KolliphorSLS fine) 1.500 1.500 Magnesium Stearate, NF (Ligamed MF-2-K) 1.5001.500 Isopropyl Alcohol, USP* — — Core Tablet 150.00 150.00 Opadry White03F180004 6.000 6.000 Purified Water* — — Isopropyl alcohol* — — TotalWeight (mg) 156.00 156.00 USP = United States Pharmacopeia; NF =National Formulary *Evaporates during the process **Weights of Compound1 hydrochloride include correction factors for purity and hydrogenchloride content of API such that the weights of Compound 1 free base in5 mg and 20 mg tablets are 5 mg and 20 mg per tablet, respectively.

The formulations provided in Table 13 further comprise inactiveingredients as provided below in Table 14. Each excipient is within thepotency limits listed for an oral route of administration in the mostcurrent FDA Inactive Ingredient Guide (IIG) as applicable. Table 15provides the quantitative composition of Opadry White 03F180004.

TABLE 14 Amount per Tablet (mg) IIG limits Component Components 5 mg 20mg (Mg) Function Lactose Monohydrate, 116.704 100.516 587.44 Diluent NF(Modified Spray dried fast Flo) Hydroxypropyl 4.50 4.50 240 BinderCellulose, NF (Klucel ELF Pharm) Croscarmellose Sodium, 9.00 9.00 180Disintegrant NF (AC-DI-SOL) Vitamin E, USP (dl-α- 0.15 0.15 1.34Antioxidant Tocopherol) Microcrystalline 11.25 11.25 234.6 DiluentCellulose, NF (Vivapur Type 101) Sodium Lauryl Sulfate, 1.50 1.50 51.69Wetting NF (Kolliphor SLS fine) agent Magnesium Stearate, NF 1.50 1.50400.748 Lubricant (Ligamed MF-2-K) Isopropyl Alcohol, USP* — — —Granulation solvent Opadry White 6.00 6.00 See Table 3 Aesthetic03F180004 for the break Coating down system Purified Water * — — —Coating solvent Isopropyl Alcohol, USP* — — — Coating solvent

TABLE 15 Blend Amount Ingredients/ IIG limits formula (mg/tablet)Compendial Reference (mg) (% w/w) 5 mg 20 mg HPMC 2910/Hypromellose92.794 60.00 3.6 3.6 (USP, PhEur, JP) Titanium Dioxide 35.7 20.00 1.21.2 (USP, FCC, PhEur, JP) Talc (USP, FCC, PhEur, JP) 220.4 10.00 0.6 0.6Macrogol/PEG (NF, FCC, 450 10.00 0.6 0.6 JECFA, Ph. Eur) MW 6000 Totalweight of Opadry White 03F180004 per tablet 6 mg 6 mg

The above-described tablets are packaged into high density polyethylene(HDPE) bottles with induction seals and packed with 1 g silica geldesiccant canisters, closed with child-resistant polypropylene screwcaps.

Example 3 Evaluation of Clinical Activity, Safety and Tolerability ofCompound 1, a Dual SYK/JAK Inhibitor, in Patients with Moderate toSevere Atopic Dermatitis

Example 3 evaluates the safety, tolerability and efficacy of Compound 1in subjects with moderate to severe atopic dermatitis, as well as thepharmacokinetic (PK) profile of Compound 1 andpharmacodyncamic/biomarkers for evidence of drug activity.

Methods and Study Design:

The study conducted was a randomized, double-blind, placebo-controlled,multicenter, sequential dose escalation study in subjects withmoderate-to severe atopic dermatitis. The study included a screeningperiod (up to 30 days) and a treatment period for 4 weeks with a 14 dayfollow up period that concluded with an end-of-study visit. Threesequential cohorts of 20, 40 and 80 mg QD were evaluated. At each doselevel a total of 12 subjects were enrolled with 9 subjects receivingCompound 1, and 3 subjects receiving matching placebos.

A total of approximately 36 subjects were randomized at approximately 10study sites in the U.S. and Canada. Dose escalation occurred after areview of the blinded safety data by a Safety Review Committee (SRC).Dose escalation continues until the Maximum Tolerated Dose (MTD) isdefined. The dose at which study drug related adverse events within thesame organ class results in treatment discontinuation in ≥2 of thesubjects (or ≥3 subjects in any system organ class), is considered toexceed the Maximum Tolerated Dose (MTD).

The dose level immediately below is considered the MTD. All data up toand including the assessments at the end of the 28-day treatment period(Day 29) of the current cohort were included in the review. The SRCreviews the blinded safety data and recommends initiation of the nextdose cohort or halting dose escalation. Lower or intermediate doselevels and alternate dosing schedules other than those proposed, may beexplored as supported by the clinical data of the previous cohort(s) inan effort to better define the MTD. Higher dose levels may be evaluatedas supported by the emerging clinical data.

Upon signing the informed consent, the each subject underwent screeningassessments from Day −30 to Day −1 prior to study drug administration.On Day 1 (baseline), eligible subjects were randomized, subjected to theDay 1/baseline assessments and received Compound 1 at 20, 40 or 80 mg orplacebo, dependent on cohort and randomization schedule. At this visit,PK/PD samples were collected at predose and up to 8 hours (or up to 12hours at selected sites). Subjects were monitored in clinic for 2 hoursfollowing the first study drug administration. Subjects returned to theclinic on Day 2 for PK and PD samples (24 hours post dose).

Pre-dose PK and PD samples were collected on Days 8 and 29 (last day oftreatment). On Day 15, PK/PD samples were also collected at pre-dose andup to 8 hours post-dose and subjects returned on Day 16 for PK and PDsamples (24 hours post dose). Subjects came in for additional safetyassessments on Days 8, 22 and 29, as well as at the end of the follow upperiod (Day 43). Disease assessments were conducted on Days 1, Day 15and Day 29.

FIG. 5 is a graphical illustration of the study design, including thetreatment period, safety follow-up period, starting doses and number ofsubjects. In the study design shown in FIG. 5, 9 active and 3 placebosubjects were randomized in each Cohort. FIG. 6 is a graphicalillustration of the patient demographics. In the demographics shown inFIG. 6, there were no use of topical or systemic steroids, and emollientuse was required with consistent application (once daily or twicedaily).

Diagnosis and Subject Inclusion/Exclusion Criteria:

Inclusion Criteria:

-   -   1. Ability to provide written informed consent obtained prior to        any study-related procedure being performed.    -   2. Male or female, 18≤years and ≤75 years of age.    -   3. Chronic AD diagnosed by the Hanifin and Rajka criteria that        has been present for at least 6 months before the screening        visit (information obtained from medical chart or patient        history).    -   4. Eczema Area and Severity Index (EASI) score ≥16 at baseline        visits.    -   5. Investigator's Global Assessment (IGA) score ≥3 at the        baseline visits.    -   6. At least 10% body surface area (BSA) of AD involvement at the        baseline visits.    -   7. Subject has a body mass index (BMI)≤35 kg/m².    -   8. History of inadequate response to topical corticosteroids or        calcineurin inhibitors as treatment for AD within 1 year before        the screening visit (information obtained from medical chart or        patient history).    -   9. Subjects must apply stable doses of an Investigator approved,        basic bland emollient once or twice-daily for at least 7 days        before the baseline visit.    -   10. Subjects must be willing to use medically effective methods        of birth control, if of reproductive potential* and sexually        active (unless they are exclusively sexually active with        same-sex partners). Adequate birth control is defined as        agreement to consistently practice an effective and accepted        method of contraception from at least 4 weeks prior to baseline        (Day 1) throughout the duration of the study and for 4 weeks        after last dose of study drug:        -   a. For females, adequate birth control methods are defined            as: hormonal contraceptives, intrauterine device (IUD),            vasectomized partner or double barrier contraception (i.e.,            condom+diaphragm, condom or diaphragm+spermicidal gel or            foam).        -   b. For males, adequate birth control methods are defined as:            vasectomy, double barrier contraception (i.e.,            condom+diaphragm, condom or diaphragm+spermicidal gel or            foam) or used by the sole partner of a hormonal            contraceptive or IUD.            -   For females, menopause is defined as 24 months without                menses; if in question, a follicle-stimulating hormone                confirming the nonchildbearing potential (refer to                laboratory reference ranges for confirmatory level) must                be documented prior to baseline visit. Hysterectomy,                bilateral oophorectomy, bilateral salpingectomy, or                bilateral tubal ligation must be documented, as                applicable.    -   11. Females of reproductive potential must have a negative serum        pregnancy test at screening and negative urine pregnancy test at        baseline (Day 0).    -   12. Subjects must be willing and able to comply with clinic        visits and study-related procedures

Exclusion Criteria:

-   -   1. Subjects have clinically infected atopic dermatitis.    -   2. Presence of any of the following laboratory abnormalities at        the screening visit:        -   a. Hemoglobin <11 g/dL        -   b. White blood cell (WBC)<3.0×10³/L        -   c. Platelet count <125×10³/L        -   d. Neutrophils <2.50×10³/L        -   e. Lymphocytes ≤1.2×10³/L        -   f. Aspartate aminotransferase (AST)/alanine aminotransferase            (ALT) >1.5× the upper limit of normal (ULN)        -   g. Total bilirubin >ULN (except for elevated indirect            bilirubin secondary to Gilbert's syndrome)        -   h. Creatinine >ULN.    -   3. Subjects with uncontrolled hypertension within the last 1        month prior to screening or blood pressure at screening of        systolic blood pressure >140 mm Hg or diastolic BP >90 mm Hg,        confirmed by repeat assessments.    -   4. Positive QuantiFERON®-TB test indicating possible        tuberculosis infection, unless there is documented evidence of a        completed adequate treatment course for latent TB.    -   5. History of latent or active tuberculosis or exposure to        endemic areas within 8 weeks.    -   6. Availability of chest radiograph at screening or within 3        months before the screening visit (radiology report must be        available) with results consistent with prior TB infection        (including but not limited to apical scarring, apical fibrosis,        or multiple calcified granuloma). This does not include        non-caseating granulomata. Screening chest radiography is not        mandatory if no clinical signs or symptoms indicating active TB        infection, unless history of latent or active tuberculosis or        exposure to endemic areas within the last 12 months.    -   7. Positive hepatitis B core antigen, positive hepatitis B        surface antigen, positive hepatitis C antibody, and/or positive        human immunodeficiency virus at the screening visit.    -   8. Subject has used hydroxyzine or diphenhydramine within 1 week        prior to baseline (Day 1).    -   9. Subject has used topical products containing urea within 1        week prior to baseline (Day 1).    -   10. Subject has used systemic antibiotics within 2 weeks or        topical antibiotics within 1 week prior to baseline (Day 1).    -   11. Subject has used any topical medicated treatment for atopic        dermatitis within 2 weeks prior to baseline (Day 1), including,        but not limited to, topical corticosteroids, calcineurin        inhibitors, tars, bleach, antimicrobials, medical devices, and        bleach baths.    -   12. Subject has used systemic treatments (other than biologics)        that could affect atopic dermatitis less than 4 weeks prior to        baseline (Day 1) (e.g., retinoids, calcineurin inhibitors,        methotrexate, cyclosporine, hydroxycarbamide [hydroxyurea],        azathioprine, oral/injectable corticosteroids). Note: Intranasal        corticosteroids, eye drops containing corticosteroids, and        inhaled corticosteroids for stable medical conditions are        allowed if subject has been on a stable dose for at least 4        weeks prior to baseline (Day 1) and will continue usage at the        same dose for the duration of the study.    -   13. Subject has received any marketed or investigational        biological agent within 12 weeks or 5 half-lives (whichever is        longer) prior to baseline (Day 1).    -   14. Subject is currently receiving a non-biological        investigational product or device or has received one within 4        weeks prior to baseline (Day 1).    -   15. Subject has excessive sun exposure, is planning a trip to a        sunny climate, or has used tanning booths within 4 weeks prior        to baseline (Day 1), or is not willing to minimize natural and        artificial sunlight exposure during the study. Use of sunscreen        products and protective apparel are recommended when exposure        cannot be avoided.    -   16. Subject has received a live attenuated vaccine within 4        weeks prior to baseline (Day 1) or plans to receive a live        attenuated vaccine during the study and up to 4 weeks or 5        half-lives (of the study product), whichever is longer, after        the last study product administration.    -   17. Subject is known to have immune deficiency or is        immunocompromised.    -   18. History of malignancy within 5 years before the baseline        visit, with the following exceptions:        -   a. subjects with a history of completely treated carcinoma            in situ of cervix, and non-metastatic squamous or basal cell            carcinoma of the skin are allowed.    -   19. Planned major surgical procedure during the length of the        patient's participation in this study.    -   20. History of congestive heart failure New York Heart        Association (NYHA) class III or IV    -   21. 12-Lead electrocardiogram (ECG) abnormalities considered by        the investigator to be clinically significant or QTc F≥450        milliseconds, regardless of clinical significance, at screening.        Abnormal ECG may be confirmed with one repeat assessment. For        subjects with QTcF≥450 msec on initial ECG, the mean of the two        QTc F assessments will determine eligibility.    -   22. Myocardial infarction, angioplasty, or cardiac stent        placement within the last 6 months.    -   23. A medical condition requiring the therapeutic use of        anticoagulants NSAID (Nonsteroidal Antiinflammatory Drugs) and        low-dose aspirin will be not considered antiplatelets.    -   24. History of hypertrophic scarring or keloid formation in        scars or suture sites.    -   25. Has difficulty swallowing medications, or known history of        malabsorption syndrome.    -   26. History of recurrent GERD (Gastroesophageal reflux disease)        requiring the use of proton pump inhibitors within the last        month.    -   27. Known history of diverticulitis.    -   28. Uncontrolled diabetes.    -   29. Any medical or psychiatric condition which, in the opinion        of the investigator or the sponsor's medical monitor, would        place the patient at risk, interfere with participation in the        study, or interfere with the interpretation of study results.    -   30. Pregnant or breast-feeding women.    -   31. Known hypersensitivity to Compound 1 or its excipients.    -   32. Prior treatment with SYK or JAK inhibitors for which the        subject received no clinical benefit, or the subject relapsed        whilst on therapy.

Investigational Product, Dosage and Mode of Administration:

Compound 1 was administered orally at doses of 20, 40 and 80 mg qd.Compound 1 was made available in 5-mg, 20-mg, and 50-mg strengthtablets.

Duration of Study:

The total treatment period for each patient was 4 weeks (to day 29) andthe total follow-up period for each patient was 14 days (to day 43).

Criteria for Evaluation:

Assessment of safety: Safety was assessed by AEs, vital signs, 12-leadECG, physical examination, and laboratory safety assessments.

Assessment of PK variables: The following PK parameters for Compound 1were derived from the concentration-time data of Compound 1 after thefirst and Day 15 dose administration in the fasted state, as dataallowed: Cmax, tmax, AUC0-∞, AUC0-t, AUC-24, λz, t½, CL/F and Vd/F

Assessment of efficacy variables: Preliminary efficacy was assessed bychanges in the following assessments between Day 1 (baseline) and Days15 and 29: IGA, EASI, 5-D Pruritus scale, Pruritus Numeric Rating Scale,% BSA involvement of AD, and skin microbiome analysis

Assessment of Pharmacodynamics/Biomarker Parameters:

-   -   1. Change from baseline in inflammatory markers in serum        (including immune markers and CRP)    -   2. Change from baseline in molecular skin biomarkers        (inflammatory and barrier)    -   3. Change from baseline in cellular markers (including reduction        in inflammatory cells)    -   4. Change from baseline in epidermal thickness and barrier        markers in skin biopsies.

Assessments of Efficacy Investigator's Global Assessment

The IGA is an assessment scale used in clinical studies to determineseverity of AD and clinical response to treatment based on a 5-pointscale ranging from 0 (clear) to 4 (severe) (18). The IGA score wasassessed at screening, day 1/baseline (pre-dose), and days 15, 29, 43 orearly termination.

TABLE 16 IGA assessment scale Score Category Definition 0 Clear Minor,residual discoloration; no erythema or induration/papulation; nooozing/crusting 1 Almost Trace, faint pink erythema with almost noinduration/ clear papulation; no oozing/crusting 2 Mild Faint pinkerythema with mild induration/papulation; disease no oozing/crusting 3Moderate Pink-red erythema with moderate induration/ disease papulation;there may be some oozing/crusting 4 Severe Deep/bright red erythema withsevere induration/ disease papulation; with oozing/crusting

5-D Pruritus Scale:

The 5-D Pruritus Scale is a 1-page, 5-question, validated questionnaireused in clinical trials to assess 5 dimensions of background itch:degree, duration, direction, disability, and distribution (19). Eachquestion corresponds to 1 of the 5 dimensions of itch; subjects ratetheir symptoms over the preceding 2-week period as “present” or on a 1to 5 scale, with 5 being the most affected. Subjects were subjected tothis assessment at the following visits: day 1/baseline (pre-dose), anddays 15, 29, 43 or early termination. The 5-D Pruritus Scale is providedin FIG. 3.

Pruritus Numeric Rating Scale:

The Pruritus NRS is a single-question assessment tool that is used toassess the patient's worst itch as a result of AD in the previous 12hours. Subjects complete the patient recorded outcome once daily.Patient compliance on the pruritus NRS is followed at each clinic visit.Subjects were instructed on daily reporting at the Baseline visit andare queried for compliance at every clinic visit. Subjects completed therating scale daily through the last study visit using the scale providedbelow.

Eczema Area and Severity Index:

The Eczema Area and Severity Index (EASI) is a validated measure used inclinical practice and clinical trials to assess the severity and extentof AD. Four AD disease characteristics are assessed for severity by theinvestigator or designee on a scale of “0” (None) through “3” (severe).In addition, the area of AD involvement is assessed as a percentage bybody area of head, trunk, upper and lower extremities and converted to ascore of 0 to 6. Subjects were subjected to this assessment at thefollowing visits: screening, day 1/baseline (pre-dose), and days 15, 29,43 or early termination. The EASI assessment tool is provided in FIG. 4.

Body Surface Area Involvement of Atopic Dermatitis

Body surface area affected by AD was assessed for each major section ofthe body (head, trunk, arms, and legs) and is reported as a percentageof all major body sections combined. Subjects were subjected to thisassessment at the following visits: screening, day1/baseline (pre-dose),and days 15, 29, 43 or early termination.

Skin Microbiome Analysis

Collection of skin microbiome samples is a non-invasive procedure wherea swab is passed along the lesional surface of the area of worst eczemainvolvement, and another swab is passed along a non-lesional area ofskin within 5 cm of the lesional site. Samples were collected from thesame lesional and non-lesional areas at day 1/baseline (pre-dose) anddays 29, 43 or early termination.

Assessment of Pharmacodynamic and Exploratory Biomarkers Assessment ofExploratory Markers Skin Biopsies:

For each subject, a maximum of four skin biopsies were collected duringthis study.

Two punch biopsy samples (one from lesional skin and one fromnon-lesional skin) were collected at Day 1 and one punch biopsy wascollected from the same lesional skin (outside the scar of the previousbiopsies) at Day 15 (optional for subjects) and Day 29.

Other Biomarkers:

A panel of biological markers were assessed to determine the effect ofCompound 1 on the disease process. These may include, but are notlimited to:

-   -   Serum cytokines and inflammatory markers    -   Molecular skin biomarkers (inflammatory and barrier)    -   Circulating and tissue resident cellular phenotyping    -   Epidermal thickness and barrier markers from skin biopsies    -   Other biomarkers related autoimmune or inflammatory diseases

Results:

FIG. 7A is a graph of the % of subjects to achieve EASI50 over time (Day1 to Day 29) for a placebo and Compound 1 in the doses of 20 mg, 40 mgand 80 mg. FIG. 7B is a graph of the % of subjects to achieve EASI75over time (Day 1 to Day 29) for a placebo and Compound 1 in the doses of20 mg, 40 mg and 80 mg. 3 subjects reached EASI90, 2 subjects with 100%clearance. FIGS. 8A-8C shows the improvement in EASI, IGA & BSA after 4weeks for a placebo, and Compound 1 in the doses of 20 mg, 40 mg and 80mg. FIG. 8A is a graph of the % CFB (percentage change from baseline)for EASI (decrease) for the placebo and Compound 1 in the doses of 20mg, 40 mg and 80 mg. FIG. 8B is a graph of the % CFB for BSA (bodysurface area) (decrease) for the placebo and Compound 1 in the doses of20 mg, 40 mg and 80 mg. FIG. 8C is a graph of the % CFB for IGA 0-1(Investigator's Global Assessment) for the placebo and Compound 1 in thedoses of 20 mg, 40 mg and 80 mg. FIG. 9 is a graph of day 15 plasmaconcentration for Compound 1 in the doses of 20 mg, 40 mg and 80 mg.FIG. 9 shows dose-dependent C_(max) and AUC, rapid oral absorption(T_(max) 2-4 hrs) and moderate rate of elimination, T_(1/2) of 10-14hrs, and low inter- and intra-individual variability. FIG. 10 is a chartshowing the inhibition of JAK and Syk kinase activity by Compound 1,Tofacitinib, Upadacitinib, and Baricitinib. In FIG. 10, the IC₅₀ valueswere determined in biochemical kinase assays using purified partial orfull length enzymes. FIG. 11 is a chart showing inhibition of Compound 1in JAK/STAT pathway in T cells stimulated with various cytokines. Asshown in FIG. 11, Compound 1 showed strong inhibition of JAK/STATpathway in T cells (primary) stimulated by various cytokines.

FIG. 12 is a chart showing Compound 1's inhibition of IL17 mediatedCCL20 release in keratinocytes. As shown in FIG. 12, unlike Tamatiniband Tofacitinib, Compound 1 inhibits IL-17-SYK mediated CCL20 release,from human keratinocytes, at levels similar to IL17 neutralizingantibodies (*p<0.05; *** p<0.001 compared to IL17+DMSO control (One-wayANOVA with Dunnett's multiple comparison test), and % is percentdecrease from IL17+DMSO control). In FIG. 12, each bar represents meanand SEM for 3 replicates (n=3) of a single donor. FIG. 13 is a graphshowing average weekly change in pruritus (NRS) for a placebo, andCompound 1 in the doses of 20 mg, 40 mg and 80 mg. As demonstrated inFIG. 13, Compound 1 shows early decrease in pruritis.

FIGS. 14A-C are graphs showing improvements in epidermal hyperplasia andcellular infiltrates observed as early as Day 15 for Compound 1 in thedoses of 20 mg, 40 mg and 80 mg. FIG. 14A is a graph showing improvementin skin thickness for Compound 1 in the doses of 20 mg, 40 mg and 80 mg.Reductions in total skin thickness can be observed as early as Day 15.FIG. 14B is a graph showing improvement in CD3+ cells for Compound 1 inthe doses of 20 mg, 40 mg and 80 mg. T cell infiltration into all layersof the skin is reduced with 40 and 80 mg ASN002 at Day 29. FIG. 14C is agraph showing improvement in CD11c+ cells for Compound 1 in the doses of20 mg, 40 mg and 80 mg. Myeloid DC infiltration into all layers of theskin is reduced with 40 and 80 mg ASN002 as early as Day 15. FIG. 15 isa chart showing Treatment-Emergent Adverse Events (TEAE), asdemonstrated in Example 3.

Conclusion

Compound 1 showed clear efficacy in moderate to severe AD patients.Compound 1 results in rapid symptom improvement—significant reduction inpatient reported itch was observed as early as 2nd day of treatment withCompound 1. Compound 1 was well tolerated in patients with moderate tosevere atopic dermatitis. Compound 1 once daily demonstrated predictablepharmacokinetics as evidenced by dose-dependent exposure, minimumindividual variability and accumulation. Compound 1 treatment downregulates inflammatory pathways, showing improvements in epidermalhyperplasia and cellular infiltrates as early as Day 15.

The most common adverse events were headache and nausea predominantlyreported on Day 1 associated with fasting and also reported from placebopatients. No serious infections or thromboembolic events occurred. Noclinically significant changes in chemistry lab parameters exceptasymptomatic, mild-to-moderate transient elevations of CPK wereobserved. No changes in lipid profile were observed. No clinicallysignificant changes in hematologic lab parameters including platelets,neutrophils and lymphocytes were observed. Subjects in the Compound 1treatment arms showed rapid onset and dose-related declines after 4weeks in EASI50 of 29%, 100% and 88% and EASI75 of 0%, 63% and 50% forthe 20, 40 and 80 mg cohorts respectively. Baseline EASI scores were29.0, 21.3 and 29.0, respectively. The average decreases in EASI and inPruritis Numeric Rating Scale (NRS) at week 4 for the 20, 40 and 80 mgcohorts were 21%, 79% and 70% and 15%, 47% and 71% respectively. In the80 mg cohort, reduction in itch was seen as early as Day 2, (˜45%) andimprovements were also observed in the 40 and 80 mg cohorts in IGAassessments (up to 38% reaching 0-1). These clinical improvements werealso associated with reversal of cutaneous biomarkers (cellularinfiltrates, immune and hyperplasia markers) particularly in the mid andhigh dose.

Abstract for Example 3

Background:

Dysregulation of Th2 and Th22 cytokine pathways are implicated in thepathogenesis of atopic dermatitis (AD). Compound 1 is a novel oralinhibitor of JAK and SYK signaling (including Tyk2), that diminishesproduction of Th2 and Th22 cytokines. Syk also regulates IL17R signalingin keratinocytes and keratinocyte differentiation. Objectives: Efficacy,Safety and Pharmacology of Compound 1 was evaluated inmoderate-to-severe AD patients in a Phase 1b randomized, double-blind,placebo-controlled study (NCT03139981).

Methods:

Patients were randomized 1:3 placebo or Compound 1 at 20, 40 or 80 mgonce daily for 4 weeks (n=36). Inclusion criteria were Eczema Area andSeverity Index (EASI) ≥16, body surface area (BSA) involvement ≥10% andan Investigator's Global Assessment (IGA) of ≥3 at baseline visit. Studyobjectives included safety/tolerability, efficacy and pharmacokineticmeasurements. No concomitant administration of topical corticosteroidsor other immunosuppressants was permitted during or prior to study.

Results: Compound 1 was very well tolerated at all dose levels. The mostcommon adverse events were transient, mild headache and nausea, mostlyrestricted to Day 1 of dosing. Subjects in the Compound 1 treatment armsshowed rapid onset and dose-related declines after 4 weeks in EASI50 of29%, 100% and 88% and EASI75 of 0%, 63% and 50% for the 20, 40 and 80 mgcohorts respectively. Baseline EASI scores were 29.0, 21.3 and 29.0,respectively. The average decreases in EASI and in Itch Numeric RatingScale (NRS) at week 4 for the 20, 40 and 80 mg cohorts were 21%, 79% and70% and 15%, 47% and 71% respectively. In the 80 mg cohort, reduction initch was seen as early as Day 2, (˜45%) and improvements were alsoobserved in the 40 and 80 mg cohorts in IGA assessments (up to 38%reaching 0-1). These clinical improvements were also associated withreversal of cutaneous biomarkers (cellular infiltrates, immune andhyperplasia markers) particularly in the mid and high dose.

Conclusion

This is a clinical report on safety, efficacy and effect on thepathologic lesional skin phenotype with oral JAK/SYK inhibitor Compound1 in moderate-to-severe AD. Compound 1 was very well tolerated anddemonstrated early improvements in pruritus and robust activity in EASIafter 4 weeks with associated reversal of cutaneous biomarkers ofinflammation.

Example 4 Clinical Activity, Safety and Tolerability of Compound 1, aDual SYK/JAK Inhibitor, in Patients with Non-Hodgkin's Lymphoma (NHL)

A formulation of the disclosure comprising2-(1-(4-((4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrilecomprises a potent inhibitor of Spleen Tyrosine Kinase (SYK) and JanusKinases (JAK). Pre-clinical studies indicate that the formulation haslow nM IC50s against SYK and JAK, decreases proliferation inibrutinib-resistant cell lines, and suppresses tumor growth in rodentxenograft models of NHL and other hematologic malignancies.

Methods:

Phase 1/2 clinical trial in patients with solid tumors and hematologicmalignancies evaluates escalating oral doses of the formulationdisclosed herein at 10, 20, 30, 40, 50, 75 and 100 mg BID and 80 and 120mg QD mg (NCT02440685). Phase 1 allows patients with solid tumors orhematologic malignancies; Phase 2 allows only patients with diffuselarge B-Cell lymphoma (DLBCL), follicular lymphoma (FL) or mantle celllymphoma (MCL). Endpoints include safety, tolerability,pharmacokinetics, serum markers of inflammation, and response usingRECIST or Lugano Classification System,

Results:

Thirty-eight patients have enrolled in the DLT phase at doses of 10mg-100 mg BID and at 80-120 mg QD. All patients had multiple prior linesof treatment (range: 2-8). The present formulation was well tolerated.No dose limiting adverse events have been reported at these dose levels.Most drug-related adverse events were Gr 1/2 (e.g. headache, fatigue).Steady-state systemic exposure was high (C_(max), AUC (0-12 h) andT_(1/2) at 40 mg BID were 0.7 μM, 6.3 μM·h and 18 h, respectively). Highsystemic exposure was also observed at 80 mg QD. Robust reduction ofCRP, IL-18, MIP1β, VCAM-1, TNFR2 was observed at all doses. About 50%reduction in target lesions at 3 months in a FL patient (Lugano, 6 priorlines) and stable disease and reduction of pruritus in a peripheralT-Cell lymphoma patient after 2 months (Lugano, 2 prior lines) oftreatment were observed. Treatment using the present formulationcontinues in both lymphoma patients. Formulation-induced lymphocytosis,indicative of recompartmentalization, has been observed in two recentlyenrolled patients. Accrual of patients continues. FIGS. 16A-16G provideclinical activity, safety and tolerability data for formulations of thepresent disclosure.

Conclusion

The present formulation was safe and well tolerated. Encouragingpreliminary evidence of efficacy in NHL patients was observed. MTD hasnot been reached and dose escalation continues.

While the present disclosure has been discussed in terms of certainembodiments, it should be appreciated that the present disclosure is notso limited. The embodiments are explained herein by way of example, andthere are numerous modifications, variations and other embodiments thatcan be employed that would still be within the scope of the presentdisclosure.

1. A pharmaceutical formulation for treating one or more diseasescharacterized by the dysregulation of the Syk/JAK pathway, comprisinggranules, wherein the granules comprise: micronized active ingredient;one or more granulation binders; one or more fillers; one or moredisintegrants; and one or more antioxidants, and the active ingredientis a compound of Formula (I) or a pharmaceutically acceptable salt,enantiomer or prodrug thereof,

wherein R1 is a 6-membered ring of Formula (II):

wherein R3 is H, OH, C(O)OH, C1 to C6 alkyl or (C1 to C6) alkyl CN; andwherein R2 is a benzene ring of Formula (III):

wherein R4 is a 6-membered ring of Formula (IV):

wherein R5 is N or CH and R6 is a hydroxyl group, methanol methyl group,or ethanol ethyl group, and wherein the formulation has a total APIdegradation impurity level not above about 0.6% of the total activeingredient amount after storage at 1 week at 40° C./75% RH in an opencontainer.
 2. The formulation of claim 1, wherein the one or moreantioxidants comprise at least one of vitamin E or butylatedhydroxytoluene.
 3. The formulation of claim 2, wherein the one or moreantioxidants is vitamin E.
 4. The formulation of claim 1, wherein themicronized granules have a particle size of less than about 20 microns.5. The formulation of claim 1, wherein the one or more fillers compriselactose monohydrate and the one or more distintegrants comprisecrospovidone.
 6. The formulation of claim 1, wherein one or moregranulation binders are selected from the group consisting ofpolyvinylpyrollidone and hydroxypropylcellulose.
 7. The formulation ofclaim 6, wherein the one or more granulation binders comprisepolyvinylpyrollidone having a number average molecular weight of about30,000.
 8. The formulation of claim 6, wherein the one or moregranulation binders comprise hydroxypropylcellulose having a viscosityat 25° C. of 75-150 centipoise in a 5% w/w in aqueous solution.
 9. Theformulation of claim 1, wherein the micronized granules have anisopropyl alcohol content of less than about 5000 ppm.
 10. Theformulation of claim 1, wherein the granules are compressed into atablet.
 11. The formulation of claim 1, further comprising one or moreextragranular components.
 12. The formulation of claim 11, wherein theextragranular components comprise one or more tableting fillers, one ormore disintegrants, one or more lubricants, and optionally one or moresurfactants.
 13. The formulation of claim 12, wherein the one or moretableting fillers comprise microcrystalline cellulose, the one or moredisintegrants comprise croscarmellose sodium, the one or moresurfactants comprise sodium lauryl sulfate, and the one or morelubricants comprise magnesium stearate.
 14. The formulation of claim 13,wherein the micronized granules and extragranular components arecompressed into a tablet.
 15. The formulation of claim 14, wherein themicronized granules have an isopropyl alcohol content of less than about5000 ppm.
 16. The formulation of claim 14, wherein the amount of activeingredient per tablet is between about 5 to 50 mg.
 17. The formulationof claim 14, wherein the amount of active ingredient per tablet isbetween about 20 to 80 mg.
 18. The formulation of claim 16, wherein theamount of active ingredient per tablet is about 5 mg, about 20 mg, orabout 50 mg.
 19. The formulation of claim 17, wherein the amount ofactive ingredient per tablet is about 20 mg, about 40 mg, or about 80mg.
 20. The formulation of claim 14, wherein the tablet has a hardnessof about 7 to 9 kP and a disintegration time of less than about 5minutes in 0.1 N HCl, pH 6.8 and 50 mM phosphate buffer at 37° C. 21.The formulation of claim 14, wherein the tablet comprises an aestheticcoating.
 22. The formulation of claim 21, wherein the coating iscomprising of hydroxypropylcellulose, titanium dioxide, talc andpolyethylene glycol.
 23. The formulation of claim 14, wherein the activeingredient is2-(1-(4-((4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile.24. The formulation of claim 14, wherein the active ingredient is2-(1-(4-((4-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile.25. The formulation of claim 1, wherein the disease comprises one ormore cancers.
 26. The formulation of claim 1, wherein the diseasecomprises one or more inflammatory disorders.
 27. The formulation ofclaim 26, wherein the inflammatory disorders comprise atopic dermatitis.28. A kit comprising one or more dosage forms for treating one or morediseases characterized by the dysregulation of the Syk/JAK pathway andinstructions for administering the dosage forms to a subject, whereinthe dosage forms comprise granules and extragranular componentscompressed into a tablet, and wherein: the micronized granules comprise:micronized active ingredient; one or more granulation binders; one ormore fillers; one or more disintegrants; and one or more antioxidants;the extragranular components comprise one or more tableting fillers, oneor more disintegrants, one or more lubricants, and optionally one ormore surfactants; the active ingredient is a compound of Formula (I) ora pharmaceutically acceptable salt, enantiomer or prodrug thereof,

wherein R1 is a 6-membered ring of Formula (II):

wherein R3 is H, OH, C(O)OH, C1 to C6 alkyl or (C1 to C6) alkyl CN; andwherein R2 is a benzene ring of Formula (III):

wherein R4 is a 6-membered ring of Formula (IV):

wherein R5 is N or CH and R6 is a hydroxyl group, methyl group, or ethylgroup, and wherein the formulation has a total API degradation impuritylevel not above about 0.6% of the total active ingredient amount afterstorage at 1 week at 40° C./75% RH in an open container.
 29. The kit ofclaim 27, wherein the disease comprises one or more cancers.
 30. The kitof claim 27, wherein the disease comprises one or more inflammatorydisorders.
 31. The kit of claim 30, wherein the inflammatory disorderscomprise atopic dermatitis.
 32. The kit of claim 31, wherein the dosageform comprises the active ingredient in a dose of about 20 mg to about80 mg.
 33. A dosage form for treating one or more diseases characterizedby the dysregulation of the Syk/JAK pathway, the dosage form comprisingmicronized granules and extragranular components compressed into atablet, and wherein: the micronized granules comprise: an activeingredient; one or more granulation binders; one or more fillers; one ormore disintegrants; and one or more antioxidants; the extragranularcomponents comprise one or more tableting fillers, one or moredisintegrants, one or more lubricants, and optionally one or moresurfactants; the active ingredient is a compound of Formula (I) or apharmaceutically acceptable salt, enantiomer or prodrug thereof,

wherein R1 is a 6-membered ring of Formula (II):

wherein R3 is H, OH, C(O)OH, C1 to C6 alkyl or (C1 to C6) alkyl CN; andwherein R2 is a benzene ring of Formula (III):

wherein R4 is a 6-membered ring of Formula (IV):

wherein R5 is N or CH and R6 is a hydroxyl group, methanol group, orethanol group, and wherein the formulation has a total API degradationimpurity level not above about 0.6% of the total active ingredientamount after storage at 1 week at 40° C./75% RH in an open container.34. The dosage form of claim 31, wherein the disease comprises or one ormore cancers.
 35. The dosage form of claim 31, wherein the diseasecomprises one or more inflammatory disorders.
 36. The dosage form ofclaim 35, wherein the inflammatory disorders comprise atopic dermatitis.37. The dosage form of claim 36, wherein the active ingredient is in anamount of about 20 mg to about 80 mg.
 38. A method of stabilizing apharmaceutical formulation, the formulation being useful for treatingone or more diseases characterized by the dysregulation of the Syk/JAKpathway, comprising: (a) mixing intragranular ingredients comprising anactive ingredient; one or more fillers; one or more disintegrants; andone or more antioxidants; (b) granulating the mixed intragranularingredients while adding a solution of 10% w/w of one or moregranulation binders in 99% v/v isopropyl alcohol until granules areformed; (c) drying and milling the granules to make micronized granules;wherein the active ingredient comprises a compound of Formula (I) or apharmaceutically acceptable salt, enantiomer or prodrug thereof:

wherein R1 is a 6-membered ring of Formula (II):

wherein R3 is H, OH, C(O)OH, C1 to C6 alkyl or (C1 to C6) alkyl CN; andwherein R2 is a benzene ring of Formula (III):

wherein R4 is a 6-membered ring of Formula (IV):

wherein R5 is N or CH and R6 is a hydroxyl group, methyl group, or ethylgroup, wherein the formulation has a total API degradation impuritylevel not above about 0.6% of the total active ingredient amount afterstorage at 1 week at 40° C./75% RH in an open container.
 39. The methodof claim 38, wherein the disease comprises one or more cancers.
 40. Themethod of claim 38, wherein the disease comprises one or moreinflammatory disorders.
 41. The method of claim 40, wherein theinflammatory disorders comprise atopic dermatitis.
 42. The method ofclaim 41, wherein the active ingredient is in an amount of about 20 mgto about 80 mg.
 43. A method of preparing a compressed tabletpharmaceutical formulation, the formulation used for treating one ormore diseases characterized by the dysregulation of the Syk/JAK pathway,comprising: (a) mixing intragranular ingredients comprising an activeingredient; one or more fillers; one or more disintegrants; and one ormore antioxidants; (b) granulating the mixed intragranular ingredientswhile adding a solution of 10% w/w of one or more granulation binders in99% v/v isopropyl alcohol until granules are formed; (c) drying andmilling the granules to make micronized granules; (d) mixing themicronized granules with extragranular components, the extragranularcomponents comprising one or more tableting fillers, one or moredisintegrants, one or more lubricants, and optionally one or moresurfactants; and compressing the micronized granules with extragranularcomponents into a tablet, wherein the active ingredient comprises acompound of Formula (I) or a pharmaceutically acceptable salt,enantiomer or prodrug thereof:

wherein R1 is a 6-membered ring of Formula (II):

wherein R3 is H, OH, C(O)OH, C1 to C6 alkyl or (C1 to C6) alkyl CN; andwherein R2 is a benzene ring of Formula (III):

wherein R4 is a 6-membered ring of Formula (IV):

wherein R5 is N or CH and R6 is a hydroxyl group, methyl group, or ethylgroup, and wherein the formulation has a total API degradation impuritylevel not above about 0.6% of the total active ingredient amount afterstorage at 1 week at 40° C./75% RH in an open container.
 44. The methodof claim 43, wherein the disease comprises one or more cancers.
 45. Themethod of claim 43, wherein the disease comprises one or moreinflammatory disorders.
 46. The method of claim 45, wherein theinflammatory disorders comprise atopic dermatitis.
 47. A method oftreating one or more diseases characterized by the dysregulation of theSyk/JAK pathway in a subject, comprising administering to the subject atherapeutically effective amount of an active ingredient in one or moredosage forms, wherein the dosage forms comprise micronized granules andextragranular components compressed into a tablet, and wherein: themicronized granules comprise: an active ingredient; one or moregranulation binders; one or more fillers; one or more disintegrants; andone or more antioxidants; the extragranular components comprise one ormore tableting fillers, one or more disintegrants, one or morelubricants, and optionally one or more surfactants; and the activeingredient is a compound of Formula (I) or a pharmaceutically acceptablesalt, enantiomer or prodrug thereof,

wherein R1 is a 6-membered ring of Formula (II):

wherein R3 is H, OH, C(O)OH, C1 to C6 alkyl or (C1 to C6) alkyl CN; andwherein R2 is a benzene ring of Formula (III):

wherein R4 is a 6-membered ring of Formula (IV):

wherein R5 is N or CH and R6 is a hydroxyl group, methyl group, or ethylgroup, and wherein the formulation has a total API degradation impuritylevel not above about 0.6% of the total active ingredient amount afterstorage at 1 week at 40° C./75% RH in an open container.
 48. The methodof claim 47, wherein the disease comprises one or more cancers.
 49. Themethod of claim 47, wherein the disease comprises one or moreinflammatory disorders.
 50. The method of claim 49, wherein theinflammatory disorders comprise atopic dermatitis.
 51. The method ofclaim 50 wherein the active ingredient is in an amount of about 20 mg toabout 80 mg.
 52. A method of manufacturing a pharmaceutical formulation,the formulation useful for treating one or more diseases characterizedby the dysregulation of the Syk/JAK pathway, comprising: (a) mixingintragranular ingredients comprising an active ingredient; one or morefillers; one or more disintegrants; and one or more antioxidants; (b)granulating the mixed intragranular ingredients while adding a solutionof 10% w/w of one or more granulation binders in 99% v/v isopropylalcohol until granules are formed; (c) drying and milling the granulesto make micronized granules; wherein the active ingredient comprises acompound of Formula (I) or a pharmaceutically acceptable salt,enantiomer or prodrug thereof:

wherein R1 is a 6-membered ring of Formula (II):

wherein R3 is H, OH, C(O)OH, C1 to C6 alkyl or (C1 to C6) alkyl CN; andwherein R2 is a benzene ring of Formula (III):

wherein R4 is a 6-membered ring of Formula (IV):

wherein R5 is N or CH and R6 is a hydroxyl group, methyl group, or ethylgroup, wherein the formulation has a total API degradation impuritylevel not above about 0.6% of the total active ingredient amount afterstorage at 1 week at 40° C./75% RH in an open container.
 53. The methodof claim 52, wherein the disease comprises one or more cancers.
 54. Themethod of claim 52, wherein the disease comprises one or moreinflammatory disorders.
 55. The method of claim 54, wherein theinflammatory disorders comprise atopic dermatitis.
 56. A compressedtablet comprising micronized granules and extragranular components,wherein: the tablet has a total weight of about 150 mg; the micronizedgranules comprise about 5 to 50 mg of an active ingredient, about 118.6to 736 mg of lactose monohydrate, about 4.5 mg croscarmellose sodium,about 0.15 mg vitamin E and about 4.5 mg granulation binder; and theextragranular components comprise about 11.25 mg microcrystallinecellulose, about 4.5 mg croscarmellose sodium and about 1.5 mg magnesiumstearate, and about 6 mg of an enteric coating; and the activeingredient is2-(1-(4-((4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile.